Information

How does the drug MBC effect the depolymerization of microtubules in eukaryotic cells?


I have tried to look for the mechanism of how methyl benzimidazol-2-yl-carbamate affects microtubules in eukaryotes, but what I found wasn't very useful:

  1. Quilan et al 1980 assert that it acts by inhibiting polymerization of microtubules rather than by directly depolymerizing them.
  2. In "The Cytoskeleton: A Target for Toxic Agents", it is mentioned that

… strains of Aspergillus that were resistant to the drug had a lower affinity for MBC in a binding assay, whereas super-sensitive strains had a higher affinity for the drug.

Any references would be appreciated.

In Summary: I would like to know how the drug MBC effects the depolymerization of microtubules in eukaryotic cells. I would also appreciate any scientific references.


Carbendazim inhibits microtubule polymerization by directly binding to tubulin, preventing subunits from interacting. Therefore, mytotic spindle malforms and potentially causes aneuploidy in affected cells. Mechanisms of resistance are often shown to be changes in membrane permeability to the compound.


CARBENDAZIM (addendum)


An expanded view of the eukaryotic cytoskeleton

A rich and ongoing history of cell biology research has defined the major polymer systems of the eukaryotic cytoskeleton. Recent studies have identified additional proteins that form filamentous structures in cells and can self-assemble into linear polymers when purified. This suggests that the eukaryotic cytoskeleton is an even more complex system than previously considered. In this essay, I examine the case for an expanded definition of the eukaryotic cytoskeleton and present a series of challenges for future work in this area.


INTRODUCTION

Electron cryomicroscopy (cryo-EM) is a family of methods that is used to study biological structure, from atoms to cells. To preserve a biological sample’s molecular details, the cryo-EM sample is kept free of the chemical fixation, dehydration, and heavy-metal staining that are commonly used in traditional EM. Furthermore, the sample is frozen so quickly that water molecules are immobilized in an amorphous state, which keeps the biological material “hydrated.” Once frozen, the sample must be kept colder than −135°C before and during image acquisition in a transmission electron cryomicroscope. Because biological matter is damaged rapidly by the microscope’s electron beam, cryo-EM imaging is done using a very limited electron dose. The resulting low-dose images are noisy and therefore require careful processing and interpretation. These stringent requirements ensure that the cryo-EM data represent biological structures in a minimally perturbed, lifelike state.

Two popular forms of cryo-EM are “single-particle” analysis (SPA) and electron cryotomography (cryo-ET). Structural biologists have used both these approaches to study macromolecular complexes, herein called complexes for brevity. SPA has revolutionized the structure determination of purified complexes. Because this method can routinely produce 3-D density maps in which protein side chains are resolved, SPA is a popular replacement for X-ray crystallography studies of complexes larger than ∼100 kDa. In contrast, cryo-ET is a method that generates modest ∼40 Å resolution 3-D density maps of complexes, organelles, and even cells. If multiple copies of a complex are identified, they can be analyzed by subtomogram averaging (STA). STA involves aligning and averaging multiple copies of the same complex, thereby increasing the signal-to-noise ratio and suppressing the missing-wedge artifacts (detailed later), resulting in higher-resolution maps (Asano et al., 2016 Oikonomou and Jensen, 2017 Hutchings and Zanetti, 2018 Schur, 2019).

This Perspective is written for cell biologists who are interested in learning and applying cryo-ET to their favorite eukaryotic systems. To cover a broad range of ideas, we have skipped some technical details, which are found in other literature and online training resources (Henderson, 1995 Koster et al., 1997 Frank, 2006a,b Dubochet, 2007 Penczek, 2010 Shen and Iwasa, 2018 Vos and Jensen, 2018 Rodenburg, 2019). We discuss some nonintuitive principles of cryo-ET and our expectations from current and future technologies. We also review some studies that use artificially thin samples like cryosections and cryolamellae because such samples vastly expand the range of questions that can be asked. Finally, we explore how cryo-ET can be used to study large cellular machines that defy conventional approaches.


Contents

The first known compound which binds to tubulin was colchicine, it was isolated from the autumn crocus, Colchicum autumnale, but it has not been used for cancer treatment. First anticancer drugs approved for clinical use were Vinca alkaloids, vinblastine and vincristine in the 1960s. They were isolated from extracts leaves of the Catharanthus roseus (Vinca rosea) plant at the University of Western Ontario in 1958. [1] First drug belong to the taxanes and paclitaxel, discovered in extracts from the bark of the yew tree, Taxus brevifolia, in 1967 by Monroe Wall and Mansukh Wani but, its tubulin inhibition activity was not known until 1979. Yews trees are poor source of active agents that limited the development of taxanes for over 20 years until discover the way of synthesis. [1] In December 1992 paclitaxel was approved to use in chemotherapy. [2]

Function Edit

Microtubules are the key components of the cytoskeleton of eukaryotic cells and have an important role in various cellular functions such as intracellular migration and transport, cell shape maintenance, polarity, cell signaling and mitosis. [3] They play a critical role in cell division by involving in the movement and attachment of the chromosomes during various stages of mitosis. Therefore, microtubule dynamics is an important target for the developing anti-cancer drugs. [1]

Structure Edit

Microtubules are composed of two globular protein subunits, α- and β-tubulin. These two subunits combine to form an α,β-heterodimer which then assembles in a filamentous tube-shaped structure. The tubulin hetero-dimers arrange themselves in a head to tail manner with the α-subunit of one dimer coming in contact with the β-subunit of the other. This arrangement results in the formation of long protein fibres called protofilaments. These protofilaments form the backbone of the hollow, cylindrical microtubule which is about 25 nanometers in diameter and varies from 200 nanometers to 25 micrometers in length. About 12–13 protofilaments arrange themselves in parallel to form a C-shaped protein sheet, which then curls around to give a pipe-like structure called the microtubule. The head to tail arrangement of the hetero dimers gives polarity to the resulting microtubule, which has an α-subunit at one end and a β-subunit at the other end. The α-tubulin end has negative (–) charges while the β-tubulin end has positive (+) charges. [3] The microtubule grows from discrete assembly sites in the cells called Microtubule organizing centers (MTOCs), which are a network of microtubule associated proteins (MAP). [4] [5]

Two molecules of energy rich guanosine triphosphate (GTP) are also important components of the microtubule structure. One molecule of GTP is tightly bound to the α-tubulin and is non-exchangeable whereas the other GTP molecule is bound to β-tubulin and can be easily exchanged with guanosine diphosphate (GDP). The stability of the microtubule will depend on whether the β-end is occupied by GTP or GDP. A microtubule having a GTP molecule at the β-end will be stable and continue to grow whereas a microtubule having a GDP molecule at the β-end will be unstable and will depolymerise rapidly. [4] [5]

Microtubule dynamics Edit

Microtubules are not static but they are highly dynamic polymers and exhibit two kinds of dynamic behaviors : 'dynamic instability' and 'treadmilling'. Dynamic instability is a process in which the microtubule ends switches between periods of growth and shortening. The two ends are not equal, the α-tubulin ringed (-)end is less dynamic while the more dynamic β-tubulin ringed (+) end grows and shortens more rapidly. Microtubule undergoes long periods of slow lengthening, brief periods of rapid shortening and also a pause in which there is neither growth nor shortening. [3] [5] [6] Dynamic instability is characterized by four variables: the rate of microtubule growth the rate of shortening frequency of transition from the growth or paused state to shortening (called a 'catastrophe') and the frequency of transition from shortening to growth or pause (called a 'rescue'). The other dynamic behavior called treadmilling is the net growth of the microtubule at one end and the net shortening at the other end. It involves the intrinsic flow of tubulin sub-units from the plus end to the minus end. Both the dynamic behaviors are important and a particular microtubule may exhibit primarily dynamic instability, treadmilling or a mixture of both. [6] [7]

Agents which act as inhibitors of tubulin, also act as inhibitors of cell division. A microtubule exists in a continuous dynamic state of growing and shortening by reversible association and dissociation of α/β-tubulin heterodimers at both the ends. This dynamic behavior and resulting control over the length of the microtubule is vital to the proper functioning of the mitotic spindle in mitosis i.e., cell division.

Microtubule is involved in different stages of the cell cycle. During the first stage or prophase, the microtubules required for cell division begins to form and grow towards the newly formed chromosomes forming a bundle of microtubules called the mitotic spindle. During prometaphase and metaphase this spindle attaches itself to the chromosomes at a particular point called the kinetochore and undergoes several growing and shortening periods in tuning with the back and forth oscillations of the chromosomes. In anaphase also, the microtubules attached to the chromosomes maintain a carefully regulated shortening and lengthening process. Thus the presence of a drug which can suppress the microtubule dynamics is sufficient to block the cell cycle and result in the death of the cells by apoptosis. [1] [8] [9]

Tubulin inhibitors thus act by interfering with the dynamics of the microtubule, i.e., growing (polymerization) and shortening (depolymerization). One class of inhibitors operate by inhibiting polymerization of tubulin to form microtubules and are called polymerization inhibitors like the colchicine analogues and the vinca alkaloids. They decrease the microtubule polymer mass in the cells at high concentration and act as microtubule-destabilizing agents. The other class of inhibitors operate by inhibiting the depolymerization of polymerized tubulin and increases the microtubule polymer mass in the cells. They act as microtubule-stabilizing agents and are called depolymerization inhibitors like the paclitaxel analogues. [3] These three classes of drugs seems to operate by slightly different mechanism.

Colchicine analogues blocks cell division by disrupting the microtubule. It has been reported that the β-subunit of tubulin is involved in colchicine binding. It binds to the soluble tubulin to form colchicine-tubulin complex. This complex along with the normal tubulins then undergoes polymerization to form the microtubule. However the presence of this T-C complex prevents further polymerization of the microtubule . This complex brings about a conformational change which blocks the tubulin dimers from further addition and thereby prevents the growth of the microtubule. As the T-C complex slows down the addition of new dimers, the microtubule disassembles due to structural imbalance or instability during the metaphase of mitosis. [11]

The Vinca alkaloids bind to the β-subunit of tubulin dimers at a distinct region called the Vinca-binding domain. They bind to tubulin rapidly, and this binding is reversible and independent of temperature (between 0 °C and 37 °C). In contrast to colchicine, vinca alkaloids bind to the microtubule directly. They do not first form a complex with the soluble tubulin nor do they copolymerize to form the microtubule, however they are capable of bringing about a conformational change in tubulin in connection with tubulin self-association. [6] Vinca alkaloids bind to the tubulin with high affinity at the microtubule ends but with low affinity at the tubulin sites present along the sides of the microtubule cylinder. The binding of these drugs at the high affinity sites results in strong kinetic suppression of tubulin exchange even at low drug concentration while their binding to the low affinity sites in relatively high drug concentration depolymerizes microtubules. [1]

In contrast to colchicine and vinca alkaloids, paclitaxel enhances microtubule polymerization promoting both the nucleation and elongation phases of the polymerization reaction, and it reduces the critical tubulin sub-unit concentration (i.e., soluble tubulin concentration at steady- state). Microtubules polymerized in presence of paclitaxel are extremely stable. [1] The binding mechanism of the paclitaxel mimic that of the GTP nucleotide along with some important differences. GTP binds at one end of the tubulin dimer keeping contact with the next dimer along each of the protofilament while the paclitaxel binds to one side of β-tubulin keeping contact with the next protofilament. GTP binds to unassembled tubulin dimers whereas paclitaxel binding sites are located only in assembled tubulin. The hydrolysis of GTP permits the disassembly and the regulation of the microtubule system however, the activation of tubulin by paclitaxel results in permanent stabilization of the microtubule. Thus the suppression of microtubule dynamics was described to be the main cause of the inhibition of cell division and of tumor cell death in paclitaxel treated cells. [12]

Tubulin binding molecules have gained much interest among cytotoxic agents due to its success in clinical oncology. They differ from the other anticancer drugs in their mode of action because they target the mitotic spindle and not the DNA. Tubulin binding drugs have been classified on the basis of their mode of action and binding site [4] [13] [14] as:

I. Tubulin depolymerization inhibitors Edit

a) Paclitaxel site ligands, includes the paclitaxel, epothilone, docetaxel, discodermolide etc.

II. Tubulin polymerization inhibitors Edit

a) Colchicine binding site, includes the colchicine, combrestatin, 2-methoxyestradiol, methoxy benzenesulfonamides (E7010) etc.

b) Vinca alkaloids binding site, [15] includes vinblastine, vincristine, vinorelbine, vinflunine, dolastatins, halichondrins, hemiasterlins, cryptophysin 52, etc.

Vinblastine bound to tubulin.

Colchicine bound to tubulin.

    and vincristine were isolated from the Madagascar periwinkle Catharanthus roseus. Madagascar traditionally used the vinca rosea to treat diabetes. In fact it has been used for centuries throughout the world to treat all kinds of ailments from wasp stings in India, to eye infections in the Caribbean. In the 1950s researchers began to analyse the plant and discovered that it contained over 70 alkaloids. Some were found to have effect on lower blood sugar levels and others act as hemostatics. The most interesting thing was that vinblastine and vincristine, were found to lower the number of white cells in blood. A high number of white cells in the blood indicates leukemia, so a new anti-cancer drug had been discovered. These two alkaloids bind to tubulin to prevent the cell from making the spindles that it needs to be able to divide. This is different from the action of taxol which interferes with cell division by keeping the spindles from being broken down. Vinblastine is mainly useful for treating Hodgkin's lymphoma, advanced testicular cancer and advanced breast cancer. Vincristine is mainly used to treat acute leukemia and other lymphomas. was developed under the direction of the French pharmacist Pierre Poiter, who, in 1989, obtained an initial license for the dug under the brand name Navelbine. Vinorelbine is also known as vinorelbine tartrate, the drug is a semi-synthetic analogue of another cancer-fighting drug, vinblastine. Vinorelbine is included in the class of pharmaceuticals known as vinca alkaloids, and many of its characteristics mimic the chemistry and biological mechanisms of the cytotoxic drugs vincristine and vinblastine. Vinorelbine showed promising activity against breast cancer and is in clinical trial for the treatment of other types of tumors. is a novel fluorinated vinca alkaloid currently in Phase II clinical trials, which in preclinical studies exhibited superior antitumor activity to vinorelbine and vinblastine. Vinflunine block mitosis at the metaphase/anaphase transition, leading to apoptosis. [17] Vinflunine is a chemotherapy drug used to treat advanced transitional cell bladder and urothelial tract cancer. It is also called Javlor. It is licensed for people who have already had cisplatin or carboplatin chemotherapy. 52 was isolated from the blue–green algaeNostoc sp. GSV 224. The cryptophycins are a family of related depsipeptides showing highly potent cytotoxic activity. Cryptophycin 52 was originally developed as a fungicide, but was too toxic for clinical use. Later the research was focused on treating cryptophycin as a microtubule poison, preventing the formation of the mitotic spindle. [10] Cryptophycin 52 showed high potent antimitotic activity to resist spindle microtubule dynamics. [4] As well, the interest in this drug has been further arose by the discovery that cryptophycin shows reduced susceptibility to the multidrug resistance pump, and shows no reduction of activity in a number of drug-resistant cell lines. was first isolated from Halichondria okadai, and later from the unrelated sponges Axinella carteri and Phankella carteri. Halichondrin B is a complex polyether macrolide which is synthesized and arrests cell growth at subnanomolar concentrations. [4] Halichondrin B is noncompetitive inhibitor of the binding of both vincristine and vinblastine to tubulin, suggesting the drugs bind to the vinca binding site, or a site nearby. The isolation of halichondrin B is from two unrelated genera of sponge, has led to speculate that halichondrin B is a microbial in reality, rather than sponge metabolite because sponges support a wide range of microbes. If this is the case, fermentation technologies could provide a useful supply of halichondrin B.
  • Dolastatins were isolated from the sea hareDolabella auricularia, a small sea mollusc, and thought to be the source of poison used to murder the son of Emperor Claudius of Rome in 55 A.D. Dolastatins 10 and 15 are novel pentapeptides and exhibit powerful antimitotic properties. They are cytotoxic in a number of cell lines at subnanomolar concentrations. The peptides of dolastatins 10 and 15 noncompetitively inhibit the binding of vincristine to tubulin. Dolastatin 10 is 9 times more potent than dolastatin 15 and both are more potent than vinblastin. [4] The dolastatins also enhance and stabilize the binding of colchicine to tubulin.
  • Hemiasterlins were isolated from the marine sponge, Cymbastela sp. The hemiasterlins are a family of potent cytotoxic peptides. Hemiasterlin A and hemiasterlin B show potent activity against the P388 cell line and inhibit cell division by binding to the vinca alkaloid site on tubulin. Hemiasterlin A and B exhibit stronger antiproliferative activities than both the vinca alkaloids and paclitaxel. an alkaloid prepared from the dried corns and seeds of the meadow saffron, Colchicum autumnale, is an anti-inflammatory drug that has been in continuous use for more than 3000 years. Colchicine is an oral drug, known to be used for treating acute gout and preventing acute attacks of familial Mediterranean fever (FMF). However, the use of colchicine is limited by its high toxicity in other therapies. Colchicine is known to inhibit cell division and proliferation. Early study demonstrated that colchicine disrupts the mitotic spindle. Dissolution of microtubules subsequently was shown to be responsible for the effect of colchicine on the mitotic spindle and cellular proliferation. [18] is isolated from the South African Willow, Combretum caffrum. Combretastatin is one of the simpler compounds to show antimitotic effects by interaction with the colchicine binding site of tubulin, and is also one of the most potent inhibitors of colchicine binding. [4] Combretastatin is not recognized by the multiple drug resistance (MDR) pump, a cellular pump which rapidly ejects foreign molecules from the cell. [8] Combretastatin is also reported to be able to inhibit angiogenesis, a process essential for tumor growth. Except those factors, one of the disadvantage of combretastatin is the low water solubility.
  • E7010 is the most active of sulfonamide antimitotic agent, which has been shown to inhibit microtubule formation by binding at the site of colchicines. [4][8] It is quite soluble in water as an acid salt. Methoxybenzene-sulfonamide showed good results against a wide range of tumor cells including vinca alkaloid resistant solid tumors. Results from animals studies indicated activity against colorectal, breast and lung cancer tissues. is a natural metabolite of the mammalian hormone oestradiol and is formed by oxidation in the liver. 2-methoxyestradiol is cytotoxic to several tumor cell lines, binds to the colchicine site of tubulin, inducing the formation of abnormal microtubules. 2-Methoxyestradiol exhibits potent apoptotic activity against rapidly growing tumor cells. It also has antiangiogenic activity through a direct apoptotic effect on endothelial cells. [19] , is a semi-synthetic analogue of paclitaxel, with a trade name Taxotere. Docetaxel has the minimal structure modifications at C13 side chain and C10 substitution showed more water solubility and more potency than paclitaxel. Clinical trials have shown that patients who develop hypersensitivity to paclitaxel may receive docetaxel without an allergic response. [4] was isolated from the bark of the Pacific yew tree Taxus brevifolia Nutt. (Taxaceae). Later it was also isolated from hazelnut trees (leaves, twigs, and nuts) and the fungi living on these trees but the concentration is only around 10% of the concentration in yew trees. Paclitaxel is also known as Taxol and Onxol to be an anti-cancer drug. The drug is the first line treatment for ovarian, breast, lung, and colon cancer and the second line treatment for AIDS-related Kaposi's sarcoma. (Kaposi sarcoma is a cancer of the skin and mucous membranes that is commonly found in patients with acquired immunodeficiency syndrome, AIDS). It is so effective that some oncologists refer to the period before 1994 as the "pre-taxol" era for treating breast cancer. [20] are derived from a fermenting soil bacteria, Sorangium cellulosum and it was found to be too toxic for use as an antifungal. Epothilones are microtubule stabilizing agents with a mechanism of action similar to taxanes, including suppression of microtubule dynamics, stabilization of microtubules, promotion of tubulin polymerization, and increased polymer mass at high concentrations. They induce mitotic arrest in the G2-M phase of the cell cycle, resulting in apoptosis. [1] Epothilone A and epothilone B exhibit both antifungal and cytotoxic properties. These epothilones are competitive inhibitors of the binding of paclitaxel to tubulin, exhibiting activity at similar concentrations. This finding leads to assume that the epothilones and paclitaxel adopt similar conformations in vivo. However, the epothilones are around 30 times more water-soluble than paclitaxel and more available, being easily obtained by fermentation of the parent myxobacterium and could be prepared by total synthesis. The epothilones also shows not to be recognized by multidrug resistant mechanisms, therefore it has much higher potency than paclitaxel in multidrug resistant cell lines. [8] was initially found to have immunosuppressive and antifungal activities. Discodermolide is a polyhydroxylated alketetraene lactone marine product, isolated from the Bahamian deep-sea sponge, Discodermia dissoluta, inhibited cell mitosis and induced formation of stable tubulin polymer in vitro and considered to be more effective than paclitaxel with EC50 value of 3.0μM versus 23μM. [4] The drug, a macrolide (polyhydroxylated lactone), is a member of a structural diverse class of compounds called polyketides with notable chemical mechanism of action. It stabilizes the microtubules of target cells, essentially arresting them at a specific stage in the cell cycle and halting cell division. It is a promising marine-derived candidate for treating certain cancers.

Colchicine is one of the oldest known antimitotic drugs and in the past years [ when? ] much research has been done in order to isolate or develop compounds having similar structure but high activity and less toxicity. This resulted in the discovery of a number of colchicine analogues. The structure of colchicine is made up of three rings, a trimethoxy benzene ring (ring A), a methoxy tropone ring (ring C) and a seven-membered ring (ring B) with an acetamido group located at its C-7 position. The trimethoxy phenyl group of colchicine not only helps in stabilizing the tubulin-colchicine complex but is also important for antitubulin activity in conjunction with the ring C. The 3-methoxy group increased the binding ability whereas the 1-methoxy group helped in attaining the correct conformation of the molecule. The stability of the tropone ring and the position of the methoxy and carbonyl group are crucial for the binding ability of the compound. The 10-methoxy group can be replaced with halogen, alkyl, alkoxy or amino groups without affecting tubulin binding affinity, while bulky substituents reduce the activity. Ring B when expanded showed reduced activity, however the ring and its C-7 side chain is thought to affect the conformation of the colchicine analogues rather than their tubulin binding ability. Substitution at C-5 resulted in loss of activity whereas attachment of annulated heterocyclic ring systems to ring B resulted in highly potent compound. [11]

Paclitaxel has achieved great success as an anti-cancer drug, yet there has been continuous effort to improve its efficacy and develop analogues which are more active and have greater bioavailability and specificity. The importance of C-13 substituted phenylisoserine side chain to bioactivity of paclitaxel has been known for a long time. Several replacements at the C-3' substitution have been tested. Replacement of the C-3' phenyl group with alkyl or alkyneyl groups greatly enhanced the activity, and with CF3 group at that position in combination with modification of the 10-Ac with other acyl groups increased the activity several times. Another modification of C-3' with cyclopropane and epoxide moieties were also found to be potent. Most of the analogues without ring A were found to be much less active than paclitaxel itself. The analogues with amide side chain at C-13 are less active than their ester counterpart. Also deoxygenation at position 1 showed reduced activity. Preparation of 10-α-spiro epoxide and its 7-MOM ether gave compounds having comparable cytotoxicity and tubulin assembly activity as that of paclitaxel. Substitution with C-6-α-OH and C-6-β-OH gave analogues which were equipotent to paclitaxel in tubulin assembly assay. Finally the oxetane ring is found to play an important role during interaction with tubulin. [21]

Vinblastine is a highly potent drug which also has serious side effects especially on the neurological system. Therefore, new synthetic analogues were developed with the goal of obtaining more efficient and less toxic drugs. The stereochemical configurations at C-20', C-16' and C-14' in the velbanamine portion are critical and inversion leads to loss of activity. The C-16' carboxymethyl group is important for activity since decarboxylated dimer is inactive. Structural variation at C-15'- C-20' in the velbanamine ring is well tolerated. The upper skeletal modification of vinblastine gave vinorelbine which shows comparable activity as that of vinblastine. Another analogue prepared was the difluoro derivative of vinorelbine which showed improved in vivo antitumor activity. It was discovered that fluorination at C-19' position of vinorelbine dramatically increased the in vivo activity. Most of the SAR studies involve the vindoline portion of bis-indole alkaloids because modification at C-16 and C-17 offers good opportunities for developing new analogues. The replacement of the ester group with an amide group at the C-16 resulted in the development of vindesine. Similarly replacement of the acetyl group at C-16 with L-trp-OC2H5, d-Ala(P)-(OC2H5)2, L-Ala(P)-(OC2H5)2 and I-Vla(P)-(OC2H5)2 gave rise to new analogues having anti- tubulin activity. Also it was found that the vindoline's indole methyl group is a useful position to functionalize potentially and develop new, potent vinblastine derivatives. A new series of semi-synthetic C-16 -spiro-oxazolidine-1,3-diones prepared from 17-deacetyl vinblastine showed good anti-tubulin activity and lower cytotoxicity. Vinglycinate a glycinate prodrug derived from the C-17-OH group of vinblastine showed similar antitumor activity and toxicity as that of vinblastine. [22]

Side effects Edit

    , a progressive, enduring, often irreversible tingling numbness, intense pain, and hypersensitivity to cold, beginning in the hands and feet and sometimes involving the arms and legs. [23]
  • stomatitis (ulceration of the lips, tongue, oral cavity)
  • nausea, vomiting, diarrhea, constipation, paralytic ileus, urinary retention
  • bone marrow suppression – flushing, localized skin reactions, rash (with or without) pruritus, chest tightness, back pain, dyspnea, drug fever, or chills
  • musculoskeletal effects – arthralgia and/or myalgia
  • severe weakness
  • hypotension
  • alopecia [24]

Human factors Edit

Limitations in anticancer therapy occur mainly due to two reasons because of the patient's organism, or because of the specific genetic alterations in the tumor cells. From the patient, therapy is limited by poor absorption of a drug which can lead to low concentration of the active agent in the blood and small amount delivery to the tumor. Low serum level of a drug can be also caused by rapid metabolism and excretion associated with affinity to intestinal or/and liver cytochrome P450. Another reason is the instability and degradation of the drugs in gastro-intestinal environment. Serious problem is also variability between patients what causes different bioavailability after administration equal dose of a drug and different tolerance to effect of chemotherapy agents. The second problem is particularly important in treatment elderly people. Their body is weaker and need to apply lower doses, often below therapeutic level. Another problem with anticancer agents is their limited aqueous solubility what substantially reduces absorption of a drug. Problems with delivery of drags to the tumor occur also when active agent has high molecular weight which limits tissue penetration or the tumor has large volume prevent for penetration. [3] [25]

Drug resistance Edit

Multidrug resistance is the most important limitation in anticancer therapy. It can develop in many chemically distinct compounds. Until now, several mechanisms are known to develop the resistance. The most common is production of so-called "efflux pumps". The pumps remove drugs from tumor cells which lead to low drug concentration in the target, below therapeutic level. Efflux is caused by P-glycoprotein called also the multidrug transporter. This protein is a product of multidrug resistance gene MDR1 and a member of family of ATP-dependent transporters (ATP-binding cassette). P-glycoprotein occurs in every organism and serves to protect the body from xenobiotics and is involved in moving nutrients and other biologically important compounds inside one cell or between cells. P-glycoprotein detects substrates when they enter the plasma membrane and bind them which causes activation of one of the ATP-binding domains. The next step is hydrolysis of ATP, which leads to a change in the shape of P-gp and opens a channel through which the drug is pumped out of the cell. Hydrolysis of a second molecule of ATP results in closing of the channel and the cycle is repeated. P-glycoprotein has affinity to hydrophobic drugs with a positive charge or electrically neutral and is often over-expressed in many human cancers. Some tumors, e.g. lung cancer, do not over-express this transporter but also are able to develop the resistance. It was discovered that another transporter MRP1 also work as the efflux pump, but in this case substrates are negatively charged natural compounds or drugs modified by glutathione, conjugation, glycosylation, sulfation and glucuronylation. Drugs can enter into a cell in few kinds of ways. Major routes are: diffusion across the plasma membrane, through receptor or transporter or by the endocytosis process. Cancer can develop the resistance by mutations to their cells which result in alterations in the surface of cells or in impaired endocytosis. Mutation can eliminate or change transporters or receptors which allows drugs to enter into the tumor cell. Other cause of drug resistance is a mutation in β tubulin which cause alterations in binding sites and a given drug cannot be bound to its target. Tumors also change expression isoforms of tubulin for these ones, which are not targets for antimitotic drugs e.g. overexpress βIII-tubulin. In addition tumor cells express other kinds of proteins and change microtubule dynamic to counteract effect of anticancer drugs. Drug resistance can also develop due to the interruption in therapy. [3] [5] [6] [25]

Others Edit

  • Marginal clinical efficacy – often compounds show activity in vitro but do not have antitumor activity in clinic. [26]
  • Poor water solubility of drugs which need to be dissolved in polyoxyethylated castor oil or polysorbate what cause hypersensitivity reactions. It has been suggested this solvents can also reduce delivery of the drugs to target cells. [10][27]
  • Bioavailability [28] limit – higher doses cause high toxicity and long-term use lead to cumulative neurotoxicity and hematopoietic toxicity. [10]
  • Neuropathy which is significant side effect can develop at any time in therapy and require an interruption of treatment. After symptoms have resolved therapy can be started again but the break allow tumor for develop of resistance. [16]
  • Poor penetration through the blood–brain barrier. [16]

Because of numerous adverse effect and limitations in use, new drugs with better properties are needed. Especially are desired improvements in antitumor activity, toxicity profile, drug formulation and pharmacology. [27] Currently have been suggested few approaches in development of novel therapeutic agents with better properties


RESULTS

Rotation and Translocation of the Nucleus

The nucleus of Swiss 3T3 fibroblast exhibits enhanced motion when treated with either monensin (Paddock and Albrecht-Buehler, 1986) or mechanical shear flow (Tseng et al., 2004). Using time-lapse phase contrast microscopy and a parallel-plate flow chamber, we documented nucleus motion within the cytoplasm of a single sheared Swiss 3T3 fibroblast (Figure 1A and Supplementary Movie 1). Application of shear flow caused minor displacement of the cells, but induced large excursions of the nucleus with respect to the rest of the cell (Figure 1B). To quantify the motion and shape of the nucleus, the following dynamic and morphometric parameters were monitored. First, we measured the translocation of the nucleus within the cytoplasm, δ, normalized by the equivalent radius of the nucleus (Figure 1C and Materials and Methods). Second, changes in the interdistances, λ, between the centroids of the nucleus and each nucleolus were measured as described previously (Tseng et al., 2004) to monitor changes in intranuclear integrity (Figure 1D). Third, changes in the shape factor of the nucleus, σ, were computed to measure possible deformations in the nuclear envelope (Figure 1D). Finally, the degree of nuclear rotation (NR), θ, was quantified by monitoring the centroid displacements of each nucleolus with respective to the centroid position of the nucleus (Figure 1D and Materials and Methods).

Figure 1. External mechanical shear stress enhances rigid-body movements of the nucleus in Swiss 3T3 fibroblasts. (A) Time-lapse phase contrast sequence of a Swiss 3T3 fibroblast subjected to shear flow for 40 min (shear stress 9.4 dyn/cm 2 ). In the first frame, the solid red circle indicates the initial position and shape of the nucleus. For reference, subsequent dashed red circles mark the initial position of the nucleus. The green circles indicate the instantaneous position and shape of the nucleus. Bar, 20 μm. (B) Net displacement of the nucleus (corrected for cell displacement) in the sheared fibroblast shown in A, using time-dependent coordinates of the nucleus centroid (xn, yn) and the cell centroid (xc, yc). (C) Normalized nucleus translocation (δ) of sheared cells (•, n = 7) and unsheared control cells (○, n = 5). (D) Schematic for the computation of nucleus shape factor σ (using both the apparent area, A, and perimeter, P, of the nucleus), nucleus-nucleolus interdistance λ, and nucleus rotation θ. Both λ and θ are computed using centroid positions of the nucleus and nucleoli as indicated by white dots (see Materials and Methods). (E) Changes in nucleus shape factor σ and nucleus-nucleolus interdistances λ normalized by their respective initial values, σo and λo, and distribution of nucleus rotation (θ) in control sheared fibroblasts (n = 7).

In sheared cells (n = 7), δ values were about twofold higher than in unsheared cells (n = 5, Figure 1C) after 40 min of shear flow and the nuclei of sheared cells moved ∼40% of their equivalent radii (Figure 1C). Both nucleus shape (σ) and intranuclear organization (λ) remained mostly intact throughout the course of the experiment, i.e., the nucleus of sheared cells showed enhanced movement without changing shape (Figure 1E and Supplementary Movie 1). We denote this type of motion, “rigid-body” motion. At early times, approximately a quarter of nucleus-nucleolus pairs exhibited at least 15° rotation (white and gray bars in Figure 1E). However, such NR ceased after 10 min of shear flow (black and red bars in Figure 1E). Because λ and σ values were constant during rotation, we describe this behavior as concerted nucleus rotation.

F-actin Depolymerization Does Not Affect Significantly Nucleus Motion

To investigate the mechanisms underlying rigid-body nucleus movement, we first examined the contributions of well-known major cytoskeletal structures, i.e., F-actin and MT. F-actin structure was depolymerized within Swiss 3T3 fibroblasts using varying concentrations of LA-B before shear flow (Figure 2A). Immunofluorescence of actin filament structure revealed extensive depolymerization in the lamella region for all tested LA-B concentrations, but F-actin structures around the nucleus were not replaced completely by small actin aggregates until LA-B reached a concentration of 1.6 μM (Figure 2A). At this concentration, the fibroblasts exhibited typical LA-B–treated morphology (Bershadsky et al., 1995), but the cells were too weakly anchored to the substratum to resist shear flows for an extended period of time. This was also the case for cells treated with 630 nM LA-B, although the cells were adherent for 25 min of shear flow. All nuclei of sheared cells treated with 630 nM LA-B or less (n = 10) exhibited movements similar to control cells (unpublished data). At 315 nM LA-B, cells (n = 3) were adherent for at least 40 min and continued to exhibit rigid-body nuclei movements. The plot of δ values showed that translocation of the nucleus was unaffected by LA-B treatment (Figure 2B). Fluorescence microscopy of 315 nM LA-B–treated cells revealed minor F-actin structures near the nuclei, whereas most F-actin was depolymerized in the lamella and perinuclear region (Figure 2A). Thus, F-actin cannot be dismissed entirely as a contributor to the coordinated motion of the nucleus because of minor residual F-actin structures that remains due to the nature of our assay. However, our results suggest that significant F-actin depolymerization does not affect the rigid-body movement in Swiss 3T3 fibroblasts under mechanical stress.

Figure 2. Cell treatment with the F-actin depolymerizing agent latrunculin-B causes no change in the motion of the nucleus. (A) F-actin organization in Swiss 3T3 fibroblasts treated with varying concentrations of F-actin depolymerizing agent, latrunculin-B (LA-B), for 30 min. F-actin and nuclear DNA were visualized using Alexa-488 phallodin and DAPI, respectively. F-actin depolymerization in the perinuclear region is evident for all tested LA-B concentrations. Significant F-actin depolymerization in the lamella did not occur until 315 nM. Bar, 20 μm. (B) Normalized nucleus centroid translocation of sheared control cells (black, n = 7) and LA-B–treated fibroblasts (315 nM, yellow, n = 3).

MT Disruption Affects Nucleus Morphology and Eliminates Rigid-Body NR

We next investigated the effect of MT integrity on nucleus motion due to their abundance in cells, especially around the nucleus (Lane and Allan, 1998). Cells were treated with the MT-disrupting agent nocodazole for 30 min before shear (n = 3). Cells showed spotted MT staining with no distinct MTOC (Figure 3A), while retaining an intact F-actin network (Figure 3A, inset). Phase contrast microscopy revealed a very ductile nucleus (Figure 3B and Supplementary Movie 6). Time-dependent values of σ and λ were no longer mostly within 5% of their initial value (Figure 3C). Hence, without an intact MT structure surrounding the nucleus, not only does the rigidity of the nuclear envelope become compromised, but the movements of nucleoli within the nucleus also become independent from each other. The distribution of rotation of nucleus-nucleolus pairs was also wider than in control cells, with some pairs of nucleus-nucleolus rotating as much as 30° from their starting position (Figure 3C). However, because both σ and λ vary dramatically, the rotation can no longer be considered concerted. In the absence of shear, the translocation of the nuclei of nocodazole-treated cells (n = 3) was slightly higher than in control cells (Figure 3D). In the presence of shear, the nuclei of nocodazoletreated fibroblasts (n = 3) exhibited dramatically increased translocation compared with untreated cells (Figure 3D). However, as with rotation, such translocation cannot be considered rigid-body motion because of widely varying σ and λ values. In contrast to F-actin depolymerization, MT depolymerization inside Swiss 3T3 fibroblasts causes both the intranuclear region and the nuclear membrane to become compromised, leading to enhanced nonconcerted nucleus movements.

Figure 3. The movement of the nucleus depends on the integrity of microtubules. (A) MT organization in fibroblasts treated for 30 min with 1 μg/ml nocodazole. Immunofluorescence of MT, nuclear DNA, and F-actin showed completely depolymerized MT structure with intact F-actin structure (inset). (B) Time-lapse phase contrast sequence of sheared fibroblast treated with nocodazole reveals a highly ductile nucleus. Solid and dashed colored circles indicate the initial position of the nucleus (green) and the initial positions of nucleoli (cyan and red) white arrows show the instantaneous location of the nucleoli. Bar, 20 μm. (C) Changes in nucleus shape factor σ and nucleus-nucleolus interdistances λ, normalized by their respective initial values, σo and λo, and distribution of nucleus rotation θ in nocodazole-treated sheared fibroblasts (n = 3). Wider distributions of σ/σo and λ/λo indicate changes in nucleus integrity, as well as nonconcerted movement of the nucleus. (D) Left: nucleus translocation in unsheared control cells (n = 5) and nocodazole-treated cells (n = 3). Right: nucleus translocation in sheared control cells (n = 7) and nocodazole-treated cells (n = 3).

Cdc42 Inactivation Causes Sustained NR

Next, we investigated, through inactivation of individual Rho GTPases, how reorganization, rather than bulk depolymerization, of cytoskeletal structures would affect nucleus motion. Members of the RhoGTPases family, RhoA, Rac1, and Cdc42, have all been shown previously to be involved in the reorganization of both F-actin and MT networks (Nobes and Hall, 1995, 1999 Bishop and Hall, 2000). Constructs of EGFP-fused RhoGTPases were generated to confirm positive transfection (Figure 4A, inset). Compared with control sheared cells, transfection of either EGFP:Cdc42T17N (n = 5), EGFP:RhoAT19N (n = 3), or EGFP:Rac1T17N (n = 3) into Swiss 3T3 fibroblasts caused minor changes in both σ and λ values (Supplementary Figure 1). But, transfection of EGFP: Cdc42T17N caused the nuclei of sheared fibroblasts to undergo sustained concerted NR (Figures 4B). Initially (t = 5–10 min), the nuclei of EGFP:Cdc42T17N transfected cells displayed a degree of rotation similar to control cells (Figures 1E and 4B). However, whereas the nucleus of control cells ceased to rotate after 10 min of shear flow, the nuclei of EGFP:Cdc42T17N transfected fibroblasts continued to rotate, with some of the nucleus-nucleolus pairs rotating as much as 10 times more than in control cells (black and red bars, Figure 4B). Unlike in nocodazole-treated cells, this enhanced rotation was not artificially increased by nucleus deformation because changes in λ and σ were comparable to control fibroblasts (Supplementary Figure 1). The translocation profiles of nuclei in fibroblasts transfected with EGFP: Cdc42T17N were dramatically different from those in control cells (Figures 4D). At early times (t <10 min), δ was comparable to control cells however, at t >10 min, δ increased sharply and became similar to δ observed in cells treated with nocodazole (Figure 4D). In contrast to transfection of dominant negative Cdc42, transfection of EGFP: Rac1T17N yielded morphometric (λ, σ) and dynamic parameters (θ, δ) that were similar to those found in sheared control cells (Figure 4, B and C). EGFP:RhoAT19N transfection caused a shift in the distribution of nucleus-nucleolus pairs that rotated at early time scales but did not cause any significant increase in either θ or δ (Figure 4, B and C). Together these results suggest that Cdc42, not RhoA or Rac1, is involved in regulating both rotation and translocation of the nucleus in Swiss 3T3 fibroblasts under mechanical stress.

Figure 4. Dominant negative Cdc42 causes sustained nucleus rotation and enhanced nucleus translocation in fibroblasts under shear stress. (A) Time-lapse phase contrast sequence of sheared fibroblasts transfected with either EGFP:Cdc42T17N (first row), EGFP:RhoAT19N (second row), or EGFP:Rac1T17N (third row). EGFP fluorescence indicates that cells are positively transfected (inset). Solid and dashed lines show the initial position and shape of the nucleus (green) and nucleoli (red and cyan) white arrows indicate the instantaneous location of nucleoli. Bar, 20 μm. (B) Distribution of nucleus rotations in sheared cells transfected with either EGFP:Cdc4217N (first row, n = 5), EGFP:RhoAT19N (second row, n = 3), or EGFP:Rac1T17N (third row, n = 3). Final rotation distributions are shown in red. (C) Normalized nucleus translocation in sheared cells transfected with either EGFP:Cdc42T17N (red, n = 5), EGFP:RhoAT19N (yellow, n = 3), or EGFP:Rac1T17N (green, n = 3). (D) Comparison of normalized nucleus translocation of sheared control cells (black, n = 7), in cells treated with nocodazole (blue, n = 3), and cells transfected with EGFP:Cdc42T17N (red, n = 5). Translocations of nuclei in EGFP:Cdc42T17N cells is comparable to control nuclei at early times (t <15 min), but becomes greater afterward, moving nearly as much as nocodazole-treated nuclei.

Shear Flow Promotes MTOC Repositioning in Swiss 3T3 Fibroblasts

We find that under shear flow, changes in either Cdc42 activity or MT integrity significantly affects the motion of the nucleus. Interestingly, Cdc42 and MT also play major roles in cell polarization (Hall, 1998 Johnson, 1999 Palazzo et al., 2001 Small et al., 2002 Fukata et al., 2003 Magdalena et al., 2003b). Structurally, the minus ends of MTs anchor at the MTOC, or the centrosome, which is positioned in close proximity to the nucleus in interphase fibroblasts (Lane and Allan, 1998 Magdalena et al., 2003a). Recently, it has been documented in C. elegans that there is a physical link between the centrosome and nucleus via Hook and SUN family proteins (Malone et al., 2003). Therefore, we speculated that our observed shear-induced nucleus motion could be caused by sheared-induced repositioning and polarization of the MTOC in Swiss 3T3 fibroblasts.

To determine changes in cell polarity, we used a MTOC localization assay described previously (Tzima et al., 2003). Through fluorescence microscopy of MT organization and nuclear DNA, we determined the relative positions of the MTOC with respect to the nucleus and the flow direction. By splitting the nucleus into two hemispheres— one in the direction of flow, one in the direction opposite of flow, and noting the location of the MTOC in relation to the demarcation line—the position of the MTOC was used as a marker of cell polarity (white lines and circles in Figure 5A). This standard MTOC localization assay was first carried out in unsheared cells (n = 75), for which the distribution of MTOC location was verified to be isotropic, i.e., 50:50 distribution on either side of the demarcation line (Figure 5B). Because the cells were transfected with various EGFP containing constructs, we also transfected the cells with blank EGFP plasmids and verified that the transfection process did not affect the random distribution of MTOC location (unpublished data). The cells were then sheared for 40 min and fixed for subsequent fluorescence microscopy. In sheared control cells (n = 75), the MTOC localized predominantly in the flow direction (Figure 5B). These results indicate that shear flow not only enhances nucleus motion, but also induces MTOC repositioning in Swiss 3T3 fibroblasts in the direction of flow.

Cdc42 Inactivation, Not RhoA or Rac1, Inhibits MTOC Polarization in Sheared Fibroblasts

We then investigated if inactivation of RhoGTPases would eliminate shear-induced polarization in Swiss 3T3 fibroblasts. In the absence of shear, control cells and cells transfected with either EGFP:RhoAT19N, EGFP:Rac1T17N, or EGFP:Cdc42T17N preserved an isotropic distribution of the MTOC around the nucleus (Figure 5B). When subject to shear flow, cells transfected with either EGFP:RhoAT19N or EGFP:Rac1T17N continued to exhibit a polarized MTOC distribution similar to control sheared cells (Figure 5B). However, transfection of EGFP:Cdc42T17N abolished shear-induced repositioning of the MTOC in the direction of flow (Figure 5B).

To ensure that MTOC polarization in fibroblasts is indeed regulated by the Cdc42 signaling pathway, we investigated downstream effectors of Cdc42. In MDCK and COS-7 cell lines, Cdc42 forms a complex with partitioning-defective protein, Par6, and mammalian atypical PKC, PKCζ (Joberty et al., 2000). Because polarization depends on the complex Cdc42-Par6-PKCζ in sheared endothelial cells and astrocytes (Etienne-Manneville and Hall, 2001, 2003 Tzima et al., 2003 Wojciak-Stothard and Ridley, 2003), we speculated that Cdc42 mediated MTOC reorientation in Swiss 3T3 fibroblasts through similar interactions with Par6 and PKCζ.

Swiss 3T3 fibroblasts were transfected with both wild-type and inactive forms of Par6B and PKCζ, respectively. To test for positive transfection, a blank EGFP vector was transfected along with Par6B and PKCζ vectors at a 1:10 ratio (Tzima et al., 2003). Cells showing positive transfection of Par6B and PKCζ vectors displayed green fluorescence similar to cells transfected with EGFP fused RhoGTPases (Figure 5A). Transfections of wild-type Par6B and PKCζ preserved an even MTOC distribution when the cells were unsheared and a polarized MTOC distribution when the cells were sheared (Figure 5B). Therefore, wild-type Par6B and PKCζ transfections did not alter the response of cells polarized by shear flow. However, cells transfected with kinase inactive PKCζ abolished shear-induced MTOC polarization (Figure 5B). The same results were obtained for ΔNt Par6B (Figure 5B). Together, these results suggest that inactivation of Cdc42, but not of RhoA or Rac1, abrogates sheared-induced MTOC polarization in Swiss 3T3 fibroblasts. Also, even if Cdc42 is active, without normal Par6B or PKCζ to interact with, the signal cannot be propagated downstream of Cdc42 to promote MTOC repositioning in sheared cells.


Microtubule polymerization

Nucleation

Nucleation is the event that initiates the formation of microtubules from the tubulin dimer. Microtubules are typically nucleated and organized by organelles called microtubule-organizing centres (MTOCs). Contained within the MTOC is another type of tubulin, γ-tubulin, which is distinct from the α- and β-subunits of the microtubules themselves. The γ-tubulin combines with several other associated proteins to form a lock washer-like structure known as the "γ-tubulin ring complex" (γ-TuRC). This complex acts as a template for α/β-tubulin dimers to begin polymerization it acts as a cap of the (−) end while microtubule growth continues away from the MTOC in the (+) direction.

The centrosome is the primary MTOC of most cell types. However, microtubules can be nucleated from other sites as well. For example, cilia and flagella have MTOCs at their base termed basal bodies. In addition, work from the Kaverina group at Vanderbilt, as well as others, suggests that the Golgi apparatus can serve as an important platform for the nucleation of microtubules. Because nucleation from the centrosome is inherently symmetrical, Golgi-associated microtubule nucleation may allow the cell to establish asymmetry in the microtubule network. In recent studies, the Vale group at UCSF identified the protein complex augmin as a critical factor for centrosome-dependent, spindle-based microtubule generation. It that has been shown to interact with γ-TuRC and increase microtubule density around the mitotic spindle origin.

Some cell types, such as plant cells, do not contain well defined MTOCs. In these cells, microtubules are nucleated from discrete sites in the cytoplasm. Other cell types, such as trypanosomatid parasites, have a MTOC but it is permanently found at the base of a flagellum. Here, nucleation of microtubules for structural roles and for generation of the mitotic spindle is not from a canonical centriole-like MTOC.

Polymerization

Following the initial nucleation event, tubulin monomers must be added to the growing polymer. The process of adding or removing monomers depends on the concentration of αβ-tubulin dimers in solution in relation to the critical concentration, which is the steady state concentration of dimers at which there is no longer any net assembly or disassembly at the end of the microtubule. If the dimer concentration is greater than the critical concentration, the microtubule will polymerize and grow. If the concentration is less than the critical concentration, the length of the microtubule will decrease.


Acknowledgments

Grant support: Danish Cancer Society (M. Jäättelä), the Danish National Research Foundation (M. Jäättelä), the Danish Medical Research Council (J. Nylandsted and M. Jäättelä), the Meyer Foundation (M. Jäättelä), the Novo Nordisk Foundation (M. Høyer-Hansen), the Vilhelm Pedersen Foundation (J. Nylandsted and M. Jäättelä), and the Danish Cancer Research Foundation (M. Jäättelä).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Ingrid Fossar Larsen for excellent technical assistance and Jiri Bartek, Anthony Cerami, Guido Kroemer, Beth Levine, and Christian Thomsen for invaluable research tools.


Results

Activation of new sites of growth independently of DNA replication

In exponentially growing wild-type cells, only one new site of growth is activated per cell cycle, early in G2. It has long been understood that activation of a new growth zone requires completion of DNA replication and a minimal cell size (Mitchison and Nurse, 1985), but there is some evidence (Rupes et al., 1999) suggesting that the potential to activate growth is present throughout the cell cycle. To test this possibility further, we checked whether more than one growth zone could be activated per cell cycle. We used the temperature-sensitive cell cycle mutant, cdc25-22, which at the restrictive temperature (36.5°C) blocks in G2 giving rise to elongated cells with a bipolar actin distribution. After 3 hours at 36.5°C, cells were treated with 50 μg/ml methyl-2-benzimidazole-carbamate (MBC) to depolymerize microtubules (lower panel of Fig. 1A). After 4 hours in MBC, around 80% of these cells branched (Fig. 1A,B, upper panel). As shown in Fig. 1B, only small stubs of tubulin were detectable in these cells. Actin accumulation was detected in the cell middle when the branch started to form, and all three ends of the branching cells were found to have actin patches and therefore be actively growing (Fig. 1C). Growth at all ends was confirmed by calcofluor staining (Fig. 1C). Using time-lapse movies of MBC-treated cdc25-22 cells at the restrictive temperature we estimated that the average cell length at branching was 31 μm, approximately twice the size of a bipolar fission yeast cell before mitosis. These results were confirmed using another cell cycle mutant, cdc2-33, most cells of which block early in G2. Under the same experimental conditions described for cdc25-22, 70% of cdc2-33 cells branched after MBC treatment whereas only 4% did so in control dimethylsulfoxide (DMSO)-treated cells (data not shown). Hence, elongated bipolar cells can activate a third growth zone, indicating that establishment of new growth sites can occur in G2 cells in the absence of further rounds of DNA replication.

Activation of new growth zones in bipolar G2 cells. (A) Branching cells during cdc25-22 block. Cells were blocked at 36.5°C for 1, 2 and 3 hours, prior to MBC/DMSO addition (length of block prior to MBC/DMSO addition shown in parentheses in the graph). Black arrows indicate time of drug treatment. The percentage of branched cells is an average of three experiments (unless otherwise stated) with s.e.m. values smaller than 5%. 100-200 cells were counted at each time point. (B) Brightfield images of blocked (3 hours) cells treated for 3 hours with DMSO (top left) or with 50 μg/ml MBC (top right and lower panels). Staining with TAT-1 antibody of MBC-treated cells, treated as above (bottom). (C) Cdc25-22 blocked (3 hours) cells treated with DMSO (left), MBC for 1 hour (middle) and for 3 hours (right), stained for actin (upper panel), with DAPI (middle panel) and with calcofluor (lower panel). (D) Frames from movie of cdc25-22 cdc13-YFP strains blocked at 36.5°C for 3 hours and then treated with MBC at t=0, at start of time-lapse. Frames were taken every 10 minutes, using an Axioplan Zeiss microscope. Bars, 5 μm.

Activation of new growth zones in bipolar G2 cells. (A) Branching cells during cdc25-22 block. Cells were blocked at 36.5°C for 1, 2 and 3 hours, prior to MBC/DMSO addition (length of block prior to MBC/DMSO addition shown in parentheses in the graph). Black arrows indicate time of drug treatment. The percentage of branched cells is an average of three experiments (unless otherwise stated) with s.e.m. values smaller than 5%. 100-200 cells were counted at each time point. (B) Brightfield images of blocked (3 hours) cells treated for 3 hours with DMSO (top left) or with 50 μg/ml MBC (top right and lower panels). Staining with TAT-1 antibody of MBC-treated cells, treated as above (bottom). (C) Cdc25-22 blocked (3 hours) cells treated with DMSO (left), MBC for 1 hour (middle) and for 3 hours (right), stained for actin (upper panel), with DAPI (middle panel) and with calcofluor (lower panel). (D) Frames from movie of cdc25-22 cdc13-YFP strains blocked at 36.5°C for 3 hours and then treated with MBC at t=0, at start of time-lapse. Frames were taken every 10 minutes, using an Axioplan Zeiss microscope. Bars, 5 μm.

To confirm that DNA replication is not required for growth activation, we generated bipolar G1 cells using the double mutant cdc10-129 cdc11-119. At 36.5°C, cdc10-129 cdc11-119 cells undergo a round of mitosis without septation and as a consequence block in G1 and stay bipolar. After MBC addition, 70% of cells became branched compared with only 7% in DMSO-treated cells (Fig. 2A,B). We conclude that activation of a new growth zone is independent of DNA replication and of cell cycle stage.

Minimal cell size requirement for new growth zone activation

We next tested whether cells need to be a minimal length to activate an extra growth zone. Populations of cells of different average cell length were obtained by blocking the cdc25-22 strain for different lengths of time (1, 2, 3, 4, 5 hours) before MBC addition. As shown in Fig. 1A, 50% of cells branched 5 to 6 hours after the cell cycle block had been imposed, irrespective of the time of MBC addition, which does not inhibit cell length extension (Sawin and Snaith, 2004), indicating that cell length is limiting for activation of an extra growth zone. We also tested branching efficiency in monopolar cells by adding MBC to blocked cdc10-129 cells. As shown in Fig. 2B, only 13% of cells had a branched phenotype after 5 hours in MBC, which indicates that monopolar cells do not branch efficiently. To investigate this further we examined whether an increase in monopolar cell size would result in an increase in branching efficiency. We compared normal-sized cells to blocked cdc25-22 cells that had been incubated at the restrictive temperature to double their size. The latter cells were then released to the permissive temperature of 25°C in the presence of the DNA synthesis inhibitor hydroxyurea (HU), which blocks cells in early S phase prior to NETO transition. Ninety minutes after release, cells were treated either with MBC or DMSO. After 3 hours of MBC treatment 25% of cells had a new growth zone in their middle. By contrast, only 4% of the normal-sized control cells activated a new growth zone in their middle (Fig. 2C). Taken together, these observations indicate that in the absence of microtubules a new growth zone can be activated in the middle of the cell but only once the cell has reached a critical minimal length.

Activation of new growth zones in G1- and S phase-arrested cells. (A) Images of cdc10-129 cdc11-119 cells at 36.5°C, after treatment with 50 μg/ml MBC for 3 hours: Nomarski (top) and DAPI stained (bottom). (B) Scoring of branching cdc10-129 and cdc10-129 cdc11-119 cells. Both cdc10-129 and cdc10-129 cdc11-119 cells were blocked for 3 hours before MBC treatment. Black arrow indicates drug addition. (C) Scoring of branching cdc25-22 cells. Cells were blocked for 3 hours at the restrictive temperature to block cells in G2 prior to mitosis. After 3 hours cells were released to the permissive temperature (25°C) to allow mitosis in the presence of 12 mM HU to prevent S phase to proceed. After 1.5 hours DMSO/MBC was added to the media. The effect of temperature and HU treatment was controlled using a wild-type strain. Both treatments had no effect on wild-type cells (data not shown). (D) Scoring of monopolar and bipolar cells in cdc25-22 cells blocked at 36.5°C and then released to 25°C in 12 mM HU. Bars, 5 μm.

Activation of new growth zones in G1- and S phase-arrested cells. (A) Images of cdc10-129 cdc11-119 cells at 36.5°C, after treatment with 50 μg/ml MBC for 3 hours: Nomarski (top) and DAPI stained (bottom). (B) Scoring of branching cdc10-129 and cdc10-129 cdc11-119 cells. Both cdc10-129 and cdc10-129 cdc11-119 cells were blocked for 3 hours before MBC treatment. Black arrow indicates drug addition. (C) Scoring of branching cdc25-22 cells. Cells were blocked for 3 hours at the restrictive temperature to block cells in G2 prior to mitosis. After 3 hours cells were released to the permissive temperature (25°C) to allow mitosis in the presence of 12 mM HU to prevent S phase to proceed. After 1.5 hours DMSO/MBC was added to the media. The effect of temperature and HU treatment was controlled using a wild-type strain. Both treatments had no effect on wild-type cells (data not shown). (D) Scoring of monopolar and bipolar cells in cdc25-22 cells blocked at 36.5°C and then released to 25°C in 12 mM HU. Bars, 5 μm.

If a minimal distance from a growing zone is necessary to activate growth, in monopolar cells the preferential site for growth activation should be at the new end, opposite the already growing zone. We therefore reasoned that in pre-elongated monopolar cells activation of growth at the new end could take place before S phase was completed. Hence, we repeated the experiment described above, leaving out the MBC treatment. The cdc25-22 cells were blocked for 3 hours at 36.5°C and then shifted to permissive temperature (25°C) in the presence of 12 mM HU. After 1 hour in HU 80% of cells underwent septation, and after 3 hours, when cells were still blocked in early S phase (FACS data not shown), 90% of the cells had actin patches at both ends (Fig. 2D), indicating that they had activated growth at the new end before the completion of S phase. To measure the cell size at NETO in the absence of DNA replication, we repeated the experiment above, varying the length of the block prior to shift down and HU treatment. Under these conditions we estimated that the average cell size at NETO in the absence of DNA replication was 17.2 μm, approximately 25% longer than a mitotic wild-type cell (see supplementary material Fig. S1).

We conclude that a new site of growth can be activated irrespective of S-phase completion and that the new site of growth is activated preferentially at the new end, which is furthest away from the actively growing old end.

The nucleus positions new sites of growth in the absence of microtubules

When microtubules are depolymerized in cells above the critical size, the new growth zone is positioned in the middle of the cell. DAPI staining in cdc25-22 (Fig. 1C) and cdc10-129 cdc11-119 (Fig. 2A) MBC-treated cells showed that the branch was always positioned in the vicinity of the nucleus. To confirm this correlation between nuclear position and location of the new growth zone, we performed time-lapse movies of cdc25-22 cells carrying yellow fluorescent protein (YFP)-tagged Cdc13 to label the nucleus (Decottignies et al., 2001). Cells were blocked at the restrictive temperature and filmed after the addition of MBC. As shown in Fig. 1D, the branch formed in the area of the cell overlaying the nucleus.

Nuclei determine position of branches. (A) Quantification of distance between nucleus and branch in individual cdc25-22-blocked cells 4 hours after centrifugation in the presence of MBC (black) or without centrifugation (grey). 82 cells were measured. DAPI staining of branching cdc25-22-blocked cells in MBC after centrifugation (lower panel). (B) Scoring of branching in cdc25-22- and cdc25-22 mto1Δ-blocked cells in the presence of either DMSO or MBC. Prior to treatment cells were blocked for 3 hours at 36.5°C. (C) Branching cdc11-119 cells treated with DMSO or MBC over a 5-hour time course. Cells were blocked at the restrictive temperature for 4 hours prior to drug treatment. (D) Images of DAPI-(top) and actin (bottom)-stained cdc11-119 cells blocked at 36.5°C for 4 hours and then treated with MBC for 6 hours. Bar, 5 μm.

Nuclei determine position of branches. (A) Quantification of distance between nucleus and branch in individual cdc25-22-blocked cells 4 hours after centrifugation in the presence of MBC (black) or without centrifugation (grey). 82 cells were measured. DAPI staining of branching cdc25-22-blocked cells in MBC after centrifugation (lower panel). (B) Scoring of branching in cdc25-22- and cdc25-22 mto1Δ-blocked cells in the presence of either DMSO or MBC. Prior to treatment cells were blocked for 3 hours at 36.5°C. (C) Branching cdc11-119 cells treated with DMSO or MBC over a 5-hour time course. Cells were blocked at the restrictive temperature for 4 hours prior to drug treatment. (D) Images of DAPI-(top) and actin (bottom)-stained cdc11-119 cells blocked at 36.5°C for 4 hours and then treated with MBC for 6 hours. Bar, 5 μm.

To confirm that positioning of this new growth site is directed by the nucleus rather than by the geometric middle of the cell, we generated cells with misplaced nuclei by centrifugation. Cdc25-22 cells were blocked for 3 hours at restrictive temperature and then centrifuged in the presence of 50 μg/ml MBC at 36°C, to move the nucleus away from the cell centre. Branch formation was followed for 4 hours. The position of the nucleus was assessed by DAPI staining and the distance between the middle of the nucleus and the centre of the branch measured in individual cells. The nucleus was displaced from the centre in more than 80% of cells (mean displacement of the nucleus was approximately 4.5 μm). As shown in Fig. 3A, in 77% of these cells the displaced nuclear position and branch position were less than 2 μm apart. These observations further support a role for the nucleus in positioning growth sites when microtubules are depolymerized. As MBC treatment leaves small tubulin stubs in the vicinity of the nucleus (Fig. 1B), we could not exclude a role for those stubs in establishment of polarized growth. To assess this possibility, we performed the same experiment in a cdc25-22 mto1Δ background. Mto1 promotes noncentrosomal microtubule nucleation and in mto1Δ cells microtubule nucleation is highly impaired and no interphase microtubule nucleating centres (iMTOC) are present (Sawin et al., 2004). As shown in Fig. 3B, the branching efficiency of cdc25-22 mto1Δ cells in MBC was comparable to that of cdc25-22 cells throughout the time course, suggesting that establishment of polarized growth is independent of microtubules. In mto1Δ cells the nucleus is off-centred (Sawin et al., 2004) and in cdc25-22 mto1Δ cells the branch was also off-centred together with the nucleus (data not shown).

We then tested whether the presence of multiple nuclei made cells competent to activate multiple growth sites. We used the cdc11-119 mutant, which at the restrictive temperature undergoes mitosis without intervening cytokinesis giving rise to multinucleated cells. After 4 hours at the restrictive temperature, most cdc11-119 cells have four nuclei. After MBC was added cells branched very efficiently by 2 hours in MBC, 80% of cells had one branch and by 4 hours 85% of cells had at least two branches (Fig. 3C), with some cells having four to six branches. All of these branches were in the vicinity of nuclei (Fig. 3D), and actin staining showed that they were all actively growing (Fig. 3D). We further assayed branch formation in cells with different numbers of nuclei by adding MBC to cdc11-119 cells blocked for 0, 2 and 4 hours, generating populations with an average of 1, 1.6 and 3.8 nuclei per cell, respectively. After 4 hours at the restrictive temperature in the presence of MBC, a strong correlation was observed between number of nuclei and number of branches (see supplementary material Fig. S2A). As the DNA content is also equally increased in cdc11-119-blocked cells, we checked branching in a cdc13 switch-off strain. Upon degradation of cdc13, cells re-replicate their DNA without intervening mitosis, giving rise to polyploid cells with one enlarged nucleus. Despite the increased DNA content, only one branch was observed upon MBC treatment (see supplementary material Fig. S2B).

Tea1 accumulates in the middle of the cell in the absence of microtubules. (A) Anti-Tea1 antibody staining of cdc25-22 cells blocked for 3 hours at 36.5°C and treated with DMSO (top) or MBC for 30, 45 and 60 minutes (below). Cells were fixed in TCA to allow visualization of Tea1 in the cell middle. Tea1 staining in the cell centre is lost with methanol fixation. (B) Brightfield images of cdc25-22 mid1Δ cells blocked for 3 hours at 36.5°C and then for 1 hour in 50 μg/ml MBC. Bar, 5 μm.

Tea1 accumulates in the middle of the cell in the absence of microtubules. (A) Anti-Tea1 antibody staining of cdc25-22 cells blocked for 3 hours at 36.5°C and treated with DMSO (top) or MBC for 30, 45 and 60 minutes (below). Cells were fixed in TCA to allow visualization of Tea1 in the cell middle. Tea1 staining in the cell centre is lost with methanol fixation. (B) Brightfield images of cdc25-22 mid1Δ cells blocked for 3 hours at 36.5°C and then for 1 hour in 50 μg/ml MBC. Bar, 5 μm.

We observed that the branches in the long multinucleated cells occurred in a sequential order, with the first branch appearing near the nucleus in the middle of the cell, and with subsequent branches occurring later near nuclei located more to the periphery of the cell. Centrifugation of blocked cdc11-119 cells in the presence of MBC resulted in the appearance of a first branch in 60% of the cells, which was displaced away from the cell centre near where the nuclei were clustered (see supplementary material Fig. S2C,D).

We conclude that in the absence of microtubules the position of a new growth zone is determined by the location of the nucleus, and that the final number of branches is determined by the number of nuclei within the cell rather than by ploidy. However, nuclei are not equivalent, and growth is activated preferentially near nuclei that are furthest away from other existing growth zones.

Microtubules reposition new growth zones away from the nucleus

When microtubules are present the activation of a new growth zone does not occur in the vicinity of the nucleus, suggesting that a positive factor activating growth may be transported away from the nucleus by the microtubules. One candidate for such a factor is the polarity marker Tea1 (Behrens and Nurse, 2002 Mata and Nurse, 1997). Fig. 4A shows that in the absence of microtubules Tea1 accumulates in the middle of the cell, in the vicinity of the nucleus, prior to the appearance of a branch. To obtain more direct evidence for the role of Tea1, or a component of the Tea1 polarity complex (Feierbach and Chang, 2001 Martin et al., 2005), being required for establishment of a new growth zone away from cell tips we deleted different known polarity factors in a cdc25-22 background and checked for branch formation in the presence of MBC at the restrictive temperature. As reported in Table 1, approximately 25% of cdc25-22 tea1Δ cells branch but no significant increase in branching was observed upon MBC treatment. At the end of the experiment cdc25-22 cells were not significantly longer than cdc25-22 tea1Δ cells (31.5 μm and 30 μm, respectively), excluding that the lack of branching of cdc25-22 tea1Δ cells was because of slower growth and consequently reduced cell size. As reported in Table 1, impairment of Tea3 (Arellano et al., 2002), Tea4 (Martin et al., 2005), Orb2 (Verde et al., 1998), Bud6 (Glynn et al., 2001) and Pom1 (Bahler and Pringle, 1998) function also caused a reduction in branching efficiency upon MBC addition. Deleting ssp1 did not cause any change in branching behaviour compared with the single mutant, whereas deletion of mid1 resulted in a great increase in branching efficiency. All cdc25-22 mid1Δ cells branched within 1 hour of MBC treatment as compared with 10% in control cdc25-22 cells (Table 1 and Fig. 4B).

Branching of double mutants after 4 hours in MBC

Genotype . DMSO . MBC .
cdc25-22 0 64%
cdc25-22 tea1Δ 23% 30%
cdc25-22 tea1Δkeltch 26% 28%
cdc25-22 tea4Δ 25% 23%
cdc25-22 bud6Δ 1% 8%
cdc25-22 sla2Δtalin 0 10%
cdc25-22 mid1Δ 0 100% (after 1 hour)
cdc2-33 0 55%
cdc2-33 ssp1Δ 0 53%
cdc2-33 tea3Δ 0 18%
cdc2-33 orb2-34 0 4%
cdc2-33 pom1Δ 9% 25%
Genotype . DMSO . MBC .
cdc25-22 0 64%
cdc25-22 tea1Δ 23% 30%
cdc25-22 tea1Δkeltch 26% 28%
cdc25-22 tea4Δ 25% 23%
cdc25-22 bud6Δ 1% 8%
cdc25-22 sla2Δtalin 0 10%
cdc25-22 mid1Δ 0 100% (after 1 hour)
cdc2-33 0 55%
cdc2-33 ssp1Δ 0 53%
cdc2-33 tea3Δ 0 18%
cdc2-33 orb2-34 0 4%
cdc2-33 pom1Δ 9% 25%

All cells were blocked for 3 hours at 36°C prior to drug treatment

If the factors required for polarized growth are transported by the microtubules to position a new growth zone, then varying microtubular length should alter the position of the branch. To test this possibility we performed a titration of MBC in cdc25-22-blocked cells and generated long bipolar cells that contained different lengths of microtubules. Fig. 5 shows that at higher MBC concentrations microtubules were shorter (for representative cells, see supplementary material Fig. S3), although the nucleus remained in the cell centre (see supplementary material Fig. S3), and the branch formed further away from the cell ends, supporting the proposal that the microtubule-Tea1 system positions the growth zone. The branching efficiency varied along the long cell axis, reaching a maximum in the cell middle furthest away from already growing tips, but decreasing significantly when the concentration of MBC was reduced and microtubules extended closer to the cell ends (Fig. 5C). Collectively, these observations suggest that microtubules are not required for the establishment of polarized cell growth but are necessary to position a new site of growth away from the nucleus.


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Microtubule drugs: action, selectivity, and resistance across the kingdoms of life

Microtubule drugs such as paclitaxel, colchicine, vinblastine, trifluralin, or oryzalin form a chemically diverse group that has been reinforced by a large number of novel compounds over time. They all share the ability to change microtubule properties. The profound effects of disrupted microtubule systems on cell physiology can be used in research as well as anticancer treatment and agricultural weed control. The activity of microtubule drugs generally depends on their binding to α- and β-tubulin subunits. The microtubule drugs are often effective only in certain taxonomic groups, while other organisms remain resistant. Available information on the molecular basis of this selectivity is summarized. In addition to reviewing published data, we performed sequence data mining, searching for kingdom-specific signatures in plant, animal, fungal, and protozoan tubulin sequences. Our findings clearly correlate with known microtubule drug resistance determinants and add more amino acid positions with a putative effect on drug-tubulin interaction. The issue of microtubule network properties in plant cells producing microtubule drugs is also addressed.

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