Fluorescent Tubulin Research Articles

Nicotinic acid attenuates amyloid β1-42-induced mitochondrial dysfunction and inhibits the mitochondrial pathway of apoptosis in differentiated SH-SY5Y cells

Alzheimer's disease (AD) is a chronic neurodegenerative disease characterized by progressive memory loss and behavioral disorders. The excessive accumulation of amyloid β (Aβ) and the formation of neurofibrillary tangles (NFTs) damage synaptic connections and the death of neurons. However, the underlying mechanisms of pathogenesis of AD remain unclear. Growing evidence indicates that impaired mitochondrial function may play a crucial role in the development of AD. In the current study, we investigated whether nicotinic acid (NA) could protect against amyloid β1-42-induced cytotoxicity in differentiated SH-SY5Y cells. Our results revealed the neuroprotective effects of NA on the differentiated SH-SY5Y cells treated with Aβ1-42. In detail, the 1-h pre-incubation with NA increased cell viability and lowered LDH levels. NA pre-incubation abolished Aβ1-42 treatment-associated alterations of mRNA levels of synaptic genes and enhanced the relative β3 Tubulin fluorescence intensity. NA eliminated the Aβ1-42-induced mitochondrial dysfunction by increasing the potential of mitochondrial membranes and maintaining a balance between the fusion and fission of mitochondria. Moreover, Aβ1-42 decreased mRNA levels of anti-apoptotic bcl2 and increased mRNA levels of pro-apoptotic: bim, bak, cytochrome c, and caspase 9. At the same time, the NA pre-treatment reduced Aβ1-42-dependent apoptotic death of differentiated SH-SY5Y cells.The above data suggest that NA presents a protective activity against Aβ1-42-induced cytotoxicity in differentiated SH-SY5Y cells by inhibiting the mitochondrial pathway of apoptosis and restoring the proper function of mitochondria.

Synthetic Spirocyclohexadienones as New Anti-Migratory Compounds in Triple- Negative Breast Cancer Cell Migration.

Triple-negative BC is the most aggressive type of breast cancer and its lack of responsiveness to conventional therapies requires screening of new chemical entities. Anti-migratory compounds are promising to treat metastatic cancer since they inhibit one of the main steps of the metastatic cascade. Spirocyclic compounds are non-conventional structures used as building blocks for the synthesis of biologically active molecules and considered interesting structures in the search for new targets in cancer research. Here, we evaluated the potential of eight synthetic spirocyclohexadienones as cell migration inhibitors. The anti-migratory ability of compounds was tested by wound healing and Boyden chamber approaches. Experiments in tubulin were performed by fluorescence and tubulin polymerization techniques. Finally, compounds were submitted to cell proliferation inhibition and flow cytometry assays to explore the mechanism by which they inhibit cell migration. Four compounds inhibited cell migration significantly. Analogs containing the 3,4,5-trimethoxyphenil ring at R1 position were the most potent and, thus, selected for additional experiments. Tubulin polymerization and fluorescence assays highlighted a possible binding of spirocyclohexadienones in the colchicine binding site; however, these compounds did not affect the cell cycle to the same extent as colchicine. Cell proliferation was affected and, notably, the most potent analogs induced apoptosis of tumor cells, suggesting a different mechanism by which they inhibit cell migration. We presented, for the first time, a series of eight synthetic spirocyclohexadienones with the ability to inhibit TNBC cell migration. These compounds represent a new category to be explored as anticancer agents.

Measurement of Microtubule Half-Life and Poleward Flux in the Mitotic Spindle by Photoactivation of Fluorescent Tubulin.

The study of microtubule dynamics is of utmost importance for the understanding of the mechanisms underlying mitotic fidelity. During mitosis, the microtubular cytoskeleton reorganizes to assemble a mitotic spindle necessary for chromosome segregation. Several methods, such as controlled exposure to cold, high pressure, high calcium concentration, or microtubule depolymerizing drugs, have been widely used to evaluate the dynamic properties of specific spindle microtubule populations. However, while these methods offer a qualitative approach that is sufficient to discern differences among specific spindle microtubule populations, they fall short in providing a robust quantitative picture that is sensitive enough to highlight minor differences, for example when comparing spindle microtubule dynamics in different genetic backgrounds. In this chapter we describe a detailed methodology to measure spindle microtubule dynamics using photoactivation of fluorescently tagged tubulin in living cells. This methodology allows the quantitative discrimination of the turnover of specific microtubule populations (e.g., kinetochore vs. non-kinetochore microtubules), as well as determination of microtubule poleward flux rates. These two conspicuous features of metazoan spindles must be tightly regulated to allow, on the one hand, efficient error correction, and on the other hand the satisfaction of the spindle assembly checkpoint that controls mitotic fidelity.

CLASP Facilitates Transitions between Cortical Microtubule Array Patterns.

Acentrosomal plant microtubule arrays form patterns at the cell cortex that influence cellular morphogenesis by templating the deposition of cell wall materials, but the molecular basis by which the microtubules form the cortical array patterns remains largely unknown. Loss of the Arabidopsis (Arabidopsis thaliana) microtubule-associated protein, CYTOPLASMIC LINKER ASSOCIATED PROTEIN (AtCLASP), results in cellular growth anisotropy defects in hypocotyl cells. We examined the microtubule array patterning in atclasp-1 null mutants and discovered a significant defect in the timing of transitions between array patterns but no substantive defect in the array patterns per se. Detailed analysis and computational modeling of the microtubule dynamics in two atclasp-1 fluorescent tubulin marker lines revealed marker-dependent effects on depolymerization and catastrophe frequency predicted to alter the steady-state microtubule population. Quantitative in vivo analysis of the underlying microtubule array architecture showed that AtCLASP is required to maintain the number of growing microtubule plus ends during transitions between array patterns. We propose that AtCLASP plays a critical role in cellular morphogenesis through actions on new microtubules that facilitate array transitions.

Role of Anti-tau Antibodies on Microtubule Polymerization and Stability

Tau is an intrinsically disordered protein which in neuronal cells promotes the polymerization and stability of microtubules (MT). In neurodegenerative diseases, tau undergoes post-translational modifications (PTMs), misfolds and forms toxic aggregates. Currently, tauopathies remain without a cure. Immunotherapies targeting tau protein in animal models induced clearance of tau pathology. However, the mechanisms of antibody-based inhibition of neurodegeneration remain mostly unclear. We have previously reported on the inhibition of tau aggregation in vitro by antibodies to non-phosphorylated tau441. We also have shown that phosphorylation of tau441 at Ser199 by GSK-3ß protein kinase may be inhibited by antibodies to phosphorylated tau. Here, we evaluated the polymerization of tubulin in the presence of non-phosphorylated tau and antibodies to non-phosphorylated tau [targeting epitopes in the N-terminus, R4, and C-terminus]. ELISA was used to determine antigen-antibody binding affinities. The microtubules were characterized by transmission electron microscopy. Tubulin fluorescence polymerization assay indicated that the antibodies reduced tubulin polymerization. In the presence of tau441, the tubulin polymerization was rescued even in the presence of antibodies. In addition to MT formation, the stability of paclitaxel-stabilized MT was also evaluated. Data indicate that tau protein and its antibodies play regulatory and/or competitive roles in MT formation/stability.

Fluorescent dyes and probes for super-resolution microscopy of microtubules and tracheoles in living cells and tissues.

We introduce fluorogenic tubulin probes based on the recently reported fluorescent dyes (510R, 580CP, GeR and SiR) and chemotherapy agents - taxanes (docetaxel, cabazitaxel and larotaxel). The cytotoxicity of the final probe, its staining performance and specificity strongly depend on both components. We found correlation between the aggregation efficiency (related to the spirolactonization of fluorophore) and cytotoxicity. Probe optimization allowed us to reach 29 ± 11 nm resolution in stimulated emission depletion (STED) microscopy images of the microtubule network in living human fibroblasts. Application to living fruit fly (Drosophila melanogaster) tissues highlighted two distinct structures: microtubules and tracheoles. We identified 6-carboxy isomers of 580CP and SiR dyes as markers for chitin-containing taenidia, a component of tracheoles. STED microscopy revealed correlation between the taenidia periodicity and the diameter of the tracheole. Combined tubulin and taenidia STED imaging showed close interaction between the microtubules and respiratory networks in living tissues of the insect larvae.

Monitoring Microtubule Dynamics in the Mouse Egg Using Photoactivatable-GFP-Tubulin.

Fluorescence photoactivation provides a strategy for monitoring protein kinetics within living cells. In particular, fluorescence photoactivation of a subpopulation of microtubule subunits within the spindle using photoactivatable fluorescent tubulin constructs has proven useful for assessing a variety of features of spindle microtubule dynamics, including poleward microtubule movement, microtubule depolymerization, and microtubule turnover, in various cellular settings. The current chapter describes a method for monitoring microtubule dynamics within the mouse egg spindle by photoactivation of photoactivatable-GFP-tubulin, followed by time-lapse confocal imaging.

Kinesin-5 mediated chromosome congression in insect spindles.

The microtubule motor protein kinesin-5 is well known to establish the bipolar spindle by outward sliding of antiparallel interpolar microtubules. In yeast, kinesin-5 also facilitates chromosome alignment "congression" at the spindle equator by preferentially depolymerizing long kinetochore microtubules (kMTs). The motor protein kinesin-8 has also been linked to chromosome congression. Therefore, we sought to determine whether kinesin-5 or kinesin-8 facilitates chromosome congression in insect spindles. RNAi of the kinesin-5 Klp61F and kinesin-8 Klp67A were performed separately in Drosophila melanogaster S2 cells to test for inhibited chromosome congression. Klp61F RNAi, Klp67A RNAi, and control metaphase mitotic spindles expressing fluorescent tubulin and fluorescent Cid were imaged, and their fluorescence distributions were compared. RNAi of Klp61F with a weak Klp61F knockdown resulted in longer kMTs and less congressed kinetochores compared to control over a range of conditions, consistent with kinesin-5 length-dependent depolymerase activity. RNAi of the kinesin-8 Klp67A revealed that kMTs relative to the spindle lengths were not longer compared to control, but rather that the spindles were longer, indicating that Klp67A acts preferentially as a length-dependent depolymerase on interpolar microtubules without significantly affecting kMT length and chromosome congression. This study demonstrates that in addition to establishing the bipolar spindle, kinesin-5 regulates kMT length to facilitate chromosome congression in insect spindles. It expands on previous yeast studies, and it expands the role of kinesin-5 to include kMT assembly regulation in eukaryotic mitosis.

Fluorescent tubulin polymerization assay to characterize moa of genotoxic compounds — A validation study

Fluorescent tubulin polymerization assay to characterize moa of genotoxic compounds — A validation study

Regulatory role of non‐phosphorylated tau 441 and its antibodies on tubulin polymerization

Tau protein is a microtubule (MT) associated protein which functions to promote polymerization and stability of microtubules (MT) in neuronal cells. When tau undergoes post‐translational modification, it misfolds and aggregates, forming cytotoxic species which lead to neurodegeneration. Immunotherapies against tauopathies have shown promise in animal models by inducing clearance of tau pathology. However, the exact mechanisms of antibody‐based inhibition of neurodegeneration remain unidentified. We have reported on anti‐tau antibodies to non‐phosphorylated tau441 and their inhibition of tau aggregation in vitro. We also showed that phosphorylation of tau441 at Ser199, by GSK‐3β, may be inhibited by anti‐tau phosphospecific antibodies. Here, we have evaluated polymerization of tubulin in the presence of non‐phosphorylated tau441 and anti‐tau antibodies to non‐phosphorylated tau [targeting epitopes in the N‐terminal, R4, and C‐terminal]. ELISA was used to determine antigen‐antibody binding affinities between tau, tubulin and anti‐tau antibodies. An anti‐tubulin antibody was used as a control in the polymerization and ELISA experiments. Using the tubulin fluorescence polymerization assay, we have discovered that the antibodies specific to non‐phosphorylated tau441 reduced tubulin polymerization. In the presence of non‐phosphorylated tau441, we observed successful tubulin polymerization even in the presence of anti‐tau antibodies. These studies indicate that tau plays a regulatory/competitive role in MT formationSupport or Funding InformationNIH/NIGMS/R15GM11905301

Abstract 4971: Quantitative image analysis of androgen receptor (AR) and tubulin biomarker profiles in circulating tumor cells (CTCs) from metastatic castration resistant prostate cancer (mCRPC) patients

Abstract Background: CTCs represent a real-time, liquid biopsy for analyses of a variety of molecular biomarkers that offer clinically relevant information. In prostate cancer, the AR pathway controls tumor cell growth and survival and is an important therapeutic target. AR becomes biologically active upon translocation from the cytoplasm to the nucleus in a microtubule dependent fashion, resulting in the activation of numerous genes. Real-time analysis of AR and tubulin status in CTCs may provide insight to therapy response in mCRPC patients. Methods: Androgen sensitive human prostate cancer cells, LNCaP, were treated with and without a synthetic androgen (R1881; 10 nM, 1 hr) and docetaxel, spiked into healthy donor blood, and captured using a prostate specific microfluidic device (GEDI chip), following the same protocol that we use with patient samples. Cells were fixed and stained (N-terminal AR, CD45, CK, DAPI, and tyr-tubulin) directly on the device and imaged using high resolution multiplex confocal microscopy. AR and tubulin status were analyzed using a quantitative image analysis algorithm. Nuclear AR percentage was calculated by integrating fluorescence intensity within regions defining the entire cell and nucleus. H-Score was calculated by multiplying the nuclear AR percentage by the normalized total cell AR intensity. Tubulin status and microtubule bundling were assessed using a variety of quantitative parameters including measurements of tubulin fluorescence intensity and distribution within the cell. Results: LNCaP cells in the presence of synthetic androgen exhibited higher nuclear AR percentage (p = 0.0004) and H-score (p = 0.003). Docetaxel treatment led to lower nuclear AR percentage (p = 0.04) and lower H-Score (p = 0.03). Cells treated with docetaxel exhibited higher tubulin intensity range (p = 0.008) and standard deviation (p = 0.03), consistent with docetaxel's mechanism of action. We applied these methods of quantitative image analyses to CTCs isolated from a small cohort of mCRPC patients. Single CTC image analysis revealed heterogeneity in AR status. Quantitative evidence for microtubule bundling was observed in patients receiving docetaxel chemotherapy. Conclusions: We have developed a high throughput quantitative image analysis algorithm to interrogate mCRPC specific biomarkers, such as AR and tubulin status, in single CTCs. These methods of quantitative image analyses will be prospectively validated with CTCs from mCRPC patients followed longitudinally over the course of treatment with AR inhibitors and taxanes, yielding patient specific, clinically relevant information that can guide physicians’ strategies for the clinical management of cancer. Citation Format: Daniel Worroll, Giuseppe Galletti, David M. Nanus, Scott T. Tagawa, Paraskevi Giannakakou. Quantitative image analysis of androgen receptor (AR) and tubulin biomarker profiles in circulating tumor cells (CTCs) from metastatic castration resistant prostate cancer (mCRPC) patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4971.

Isolation of Functional Tubulin Dimers and of Tubulin-Associated Proteins from Mammalian Cells

The microtubule (MT) cytoskeleton forms a dynamic filamentous network that is essential for many processes, including mitosis, cell polarity and shape, neurite outgrowth and migration, and ciliogenesis [1, 2]. MTs are built up of α/β-tubulin heterodimers, and their dynamic behavior is in part regulated bytubulin-associated proteins (TAPs). Here we describe a novel system to study mammalian tubulins and TAPs. We co-expressed equimolar amounts of triple-tagged α-tubulin and β-tubulin using a 2A "self-cleaving" peptide and isolated functional fluorescent tubulin dimers from transfected HEK293T cells with a rapid two-step approach. We also produced two mutant tubulins that cause brain malformations in tubulinopathy patients [3]. We then applied a paired mass-spectrometry-based method to identify tubulin-binding proteins in HEK293T cellsand describe both novel and known TAPs. We find that CKAP5 and the CLASPs, which are MT plus-end-tracking proteins with TOG(L)-domains[4], bind tubulin efficiently, as does the Golgi-associatedprotein GCC185, which interacts with the CLASPs [5]. The N-terminal TOGL domain of CLASP1 contributes to tubulin binding and allows CLASP1 to function as an autonomous MT-growth-promoting factor. Interestingly, mutant tubulins bind less well to a number of TAPs, including CLASPs and GCC185, and incorporate less efficiently into cellular MTs. Moreover, expression of these mutants in cells impairs several MT-growth-related processes involving TAPs. Thus, stable tubulin-TAP interactions regulate MT nucleation and growth in cells. Combined, our results provide a resource for investigating tubulin interactions and functions and widen the spectrum of tubulin-related disease mechanisms.

Designing Authentic Undergraduate Research Experiences in a Single-Semester Lab Course

The importance of a robust undergraduate research experience has been demonstrated time and again. However, too few undergraduates engage in genuine research and leverage this opportunity. Here, I present a laboratory course in cell and molecular biology that is designed to mimic a true research project. Students work through a 10-step experimental design culminating in the construction, expression, and visualization of microtubules fused to green fluorescent protein in baker's yeast. The steps of this project include the isolation of the tubulin gene from yeast genomic DNA, the cloning of that gene into an expression vector, the amplification of this plasmid in E. coli, and the expression of fluorescent tubulin in yeast. Controls and validation steps are embedded throughout the project, as they would be in a genuine research project. This laboratory course more closely resembles a one-semester undergraduate research experience than a typical lab course. However, because this course reaches a much larger number of students compared with undergraduate research opportunities, it provides students with a valuable research experience that remains confined to the scheduled time block of a typical lab course. In this way, many of the benefits of research are experienced by a large number of undergraduates.

Cellular effects of curcumin on Plasmodium falciparum include disruption of microtubules.

Curcumin has been widely investigated for its myriad cellular effects resulting in reduced proliferation of various eukaryotic cells including cancer cells and the human malaria parasite Plasmodium falciparum. Studies with human cancer cell lines HT-29, Caco-2, and MCF-7 suggest that curcumin can bind to tubulin and induce alterations in microtubule structure. Based on this finding, we investigated whether curcumin has any effect on P. falciparum microtubules, considering that mammalian and parasite tubulin are 83% identical. IC50 of curcumin was found to be 5 µM as compared to 20 µM reported before. Immunofluorescence images of parasites treated with 5 or 20 µM curcumin showed a concentration-dependent effect on parasite microtubules resulting in diffuse staining contrasting with the discrete hemispindles and subpellicular microtubules observed in untreated parasites. The effect on P. falciparum microtubules was evident only in the second cycle for both concentrations tested. This diffuse pattern of tubulin fluorescence in curcumin treated parasites was similar to the effect of a microtubule destabilizing drug vinblastine on P. falciparum. Molecular docking predicted the binding site of curcumin at the interface of alpha and beta tubulin, similar to another destabilizing drug colchicine. Data from predicted drug binding is supported by results from drug combination assays showing antagonistic interactions between curcumin and colchicine, sharing a similar binding site, and additive/synergistic interactions of curcumin with paclitaxel and vinblastine, having different binding sites. This evidence suggests that cellular effects of curcumin are at least, in part, due to its perturbing effect on P. falciparum microtubules. The action of curcumin, both direct and indirect, on P. falciparum microtubules is discussed.

Preparation of Fluorescent Tubulin Binders

AbstractInvited for this month’s cover are the groups of Prof. Daniele Passarella, Prof. Graziella Cappelletti, Dr. Stephen Faulkner and Prof. John S. Snaith based at the Universities of Milano, Oxford and Birmingham. The image shows that a fluorescent tubulin binder could evidence the tubulin/microtubule network.

The Structure of the Mitotic Spindle and Nucleolus during Mitosis in the Amebo-Flagellate Naegleria

Mitosis in the amebo-flagellate Naegleria pringsheimi is acentrosomal and closed (the nuclear membrane does not break down). The large central nucleolus, which occupies about 20% of the nuclear volume, persists throughout the cell cycle. At mitosis, the nucleolus divides and moves to the poles in association with the chromosomes. The structure of the mitotic spindle and its relationship to the nucleolus are unknown. To identify the origin and structure of the mitotic spindle, its relationship to the nucleolus and to further understand the influence of persistent nucleoli on cellular division in acentriolar organisms like Naegleria, three-dimensional reconstructions of the mitotic spindle and nucleolus were carried out using confocal microscopy. Monoclonal antibodies against three different nucleolar regions and α-tubulin were used to image the nucleolus and mitotic spindle. Microtubules were restricted to the nucleolus beginning with the earliest prophase spindle microtubules. Early spindle microtubules were seen as short rods on the surface of the nucleolus. Elongation of the spindle microtubules resulted in a rough cage of microtubules surrounding the nucleolus. At metaphase, the mitotic spindle formed a broad band completely embedded within the nucleolus. The nucleolus separated into two discreet masses connected by a dense band of microtubules as the spindle elongated. At telophase, the distal ends of the mitotic spindle were still completely embedded within the daughter nucleoli. Pixel by pixel comparison of tubulin and nucleolar protein fluorescence showed 70% or more of tubulin co-localized with nucleolar proteins by early prophase. These observations suggest a model in which specific nucleolar binding sites for microtubules allow mitotic spindle formation and attachment. The fact that a significant mass of nucleolar material precedes the chromosomes as the mitotic spindle elongates suggests that spindle elongation drives nucleolar division.

How we discovered fluorescent speckle microscopy

Fluorescent speckle microscopy (FSM) is a method for measuring the movements and dynamic assembly of macromolecular assemblies such as cytoskeletal filaments (e.g., microtubules and actin) or focal adhesions within large arrays in living cells or in preparations in vitro. The discovery of the method depended on recognizing the importance of unexpected fluorescence images of microtubules obtained by time-lapse recording of vertebrate epithelial cells in culture. In cells that were injected with fluorescent tubulin at ∼10% of the cytosol pool, microtubules typically appeared as smooth threads with a nearly constant fluorescence intensity. One day, when an unusually low concentration of fluorescent tubulin was injected into cells, the images from a sensitive cooled charge-coupled detector camera showed microtubules with an unusual “speckled” appearance—there were fluorescent dots with variable intensity and spacing along the microtubules. A first thought was that the speckles were an artifact. With further thought, we surmised that the speckles could be telling us something about stochastic association of tubulin dimers with the growing end of a microtubule. Numerous experiments confirmed the latter hypothesis. Subsequently the method we call FSM has proven to be very valuable. The speckles turned out not to be a meaningless artifact, but rather a serendipitous find.

Overexpression of puromycin sensitive aminopeptidase (PSA/NPEPPS) reduces soluble tau and delays development of neuropathology in PSA/TAU-P301L double transgenic mice

Results: We found that pseudophosphorylated tau aggregates in cells when Thr 212 is mutated to Glu (Ps-tau), suggesting that phosphorylation at this site facilitates tau self-assembly. The expression of tau pseudophosphorylated at Thr212, Thr231, and Ser262 disrupts the microtubules when co-transfected with fluorescent tubulin. EB-1 is a protein that binds to the growing end of the microtubules. Cells stably transfected with fluorescent EB-1 are used to visualize the dynamics of these filaments in the live cells. These cells were transfected with an inducible system to express tau upon withdraw of doxycycline (TET-off). The effect of Ps-tau on these cells was also studied. Conclusions: These findings suggest that tau phosphorylation at Thr 212 facilitates tau self-aggregation, and that the combination of phosphorylation at Thr212, 231 and Ser262 in the same tau molecule can trigger toxic reaction.

Abstract 3466: Increased steady-state levels of microtubules in aberrant distributions in Ni+2 ion-/MCA-transformed C3H/10T1/2 mouse embryo cell lines

Abstract We previously found amplification of the Ect-2 proto-oncogene, and higher steady-state levels of ect-2 mRNA and protein, in Ni+2 ion- and MCA-transformed C3H/10T1/2 (10T1/2) mouse embryo fibroblast cell lines. Here, we tested the hypotheses that there were 1) increased steady-state levels of microtubules in Ni+2/MCA-transformed 10T1/2 cell lines, 2) aberrant distributions of microtubules in transformed cell lines, and 3) increases in cross sectional areas of transformed cell lines. Log phase Ni+2 ion- and MCA-transformed and non-transformed 10T1/2 mouse embryo cells were stained separately with fluorescent antibodies to alpha tubulin or beta tubulin, then visualized using confocal microscopy (n=8). The number of microtubules/cell and cross sectional areas of stained cells were quantified using the volocity image analysis program, and ratios of (microtubules in transformed cell lines/microtubules in non-transformed 10T1/2 cells) were determined. In non-transformed 10T1/2 cells, microtubules were distributed homogeneously. In transformed cells, microtubules were often present as large aggregates in cells, and shapes of transformed cells were aberrant compared to shapes of non-transformed 10T1/2 cells. Compared to non-transformed 10T1/2 cells, MCA Cl 16, NiS3A1, NiS7A1 and NiO2C3 cell lines had a 2.1 (p < 0.001), 1.5 (p > 0.05), 1.8 (p > 0.05) and 1.9 (p < 0.05) fold higher fluorescent intensity for alpha tubulin, respectively. Compared to non-transformed 10T1/2 cells, MCA Cl 16, NiS3A1, NiS7A1 and NiO2C3 had 2.5 (p < 0.05) 1.7 (p<0.05), 1.8 (p>0.05) and 1.1 (p<0.05) fold-higher beta tubulin fluorescence intensity relative to 10T1/2 cells. Transformed cell lines showed 1.9 - 2.5 significant (p<0.1) fold-increases in cross-sectional area compared to non-transformed 10T1/2 cells (n = 8). We concluded MCA-/Ni+2 ion-transformed 10T1/2 cells had higher steady-sate levels of microtubules, present in aberrant distributions, and transformed cells were larger than non-transformed cells. Supported by Grant ES03341 from U. S. N. I. H., funding from the M. S. Program in the Dept. of Molecular Microbiology/Immunology at USC, and discretionary funding, to JRL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3466.

Binding affinity of fluorochromes and fluorescent proteins to Taxol™ crystals

The ability of Taxol (paclitaxel) to bind and stabilize microtubules is the basis for its use as an anti-mitotic drug as well as an additive for in vivo and in vitro studies of microtubules. The low solubility of Taxol in aqueous solutions, however, facilitates the formation of Taxol crystals that can be decorated with fluorescent tubulin. In cells treated with Taxol, these decorated Taxol crystals may be mistaken for self-assembled tubulin asters when observed with a fluorescent microscope. We confirmed via fluorescent and differential interference contrast microscopy that Taxol crystals can be decorated not only with fluorescent tubulin but also with other fluorescent proteins and fluorochromes without perturbing their morphology. We used theoretical calculations to further investigate Taxol-fluorescent agent interactions. Using computational docking studies we identified a new, potential Taxol binding site within the tubulin dimmer, allowing the interaction between crystalline Taxol and tubulin. Our calculations, however, show that fluorescent tubulin binding to Taxol crystals is more favorable via the fluorochromes covalently linked to the tubulin dimmers, rather than via the new Taxol-binding site, what is in accordance with our experimental data.