Probes for Tubulin and Other Cytoskeletal Proteins—Section 11.2

Paclitaxel

We offer paclitaxel (P3456) for research purposes only at a purity of >98% by HPLC. Paclitaxel, formerly referred to as taxol in some scientific literature, is the approved generic name for the anticancer pharmaceutical Taxol (a registered trademark of Bristol-Myers Squibb Co.). The diterpenoid paclitaxel is a potent anti-neoplastic agent originally isolated from the bark and needles of the western yew tree, Taxus brevifolia. The anti-mitotic and cytotoxic action of paclitaxel is related to its ability to promote tubulin assembly into stable aggregated structures that cannot be depolymerized by dilution, calcium ions, cold or a number of microtubule-disrupting drugs; paclitaxel also decreases the critical concentration of tubulin required for microtubule assembly. Cultured cells treated with paclitaxel are blocked in the G2 (the "gap" between DNA synthesis and mitosis) and M (mitosis) phases of the cell cycle.

TubulinTracker Green Reagent

TubulinTracker Green reagent (T34075) provides green-fluorescent staining of polymerized tubulin in live cells. Also known as Oregon Green 488 paclitaxel bis-acetate (a bi-acetylated version of Oregon Green 488 paclitaxel (P22310), see below), TubulinTracker Green reagent is an uncharged, nonfluorescent compound () that easily passes through the plasma membrane of live cells. Once inside the cell, the lipophilic blocking group is cleaved by nonspecific esterases, resulting in a green-fluorescent, charged paclitaxel.

TubulinTracker Green reagent is provided as a set of two components: lyophilized TubulinTracker Green reagent and a 20% Pluronic F-127 solution in dimethylsulfoxide (DMSO), a solubilizing agent for making stock solutions and facilitating cell loading. Please note that because taxol binds polymerized tubulin, TubulinTracker Green reagent will inhibit cell division and possibly other functions utilizing polymerized tubulin in live cells.

Fluorescent Paclitaxel Conjugates

In addition to unlabeled paclitaxel and TubulinTracker Green reagent, we provide three fluorescent derivatives of paclitaxel: Oregon Green 488 paclitaxel (Flutax-2, P22310), BODIPY FL paclitaxel (P7500) and BODIPY 564/570 paclitaxel (P7501). These fluorescent paclitaxel derivatives are promising tools for imaging microtubule formation and motility. Their fluorescent attributes should also make these conjugates useful reagents for screening compounds that affect microtubule assembly.

Oregon Green 488 paclitaxel is an important probe for labeling tubulin filaments in live cells. The fluorescent label on this probe is attached by derivatizing the 7β-hydroxy group of native paclitaxel (), a strategy that permits selective binding of the probe to microtubules with high affinity at 37°C (K_d ~10^-7 M). Oregon Green 488 paclitaxel has been utilized in a high-throughput fluorescence polarization–based assay to screen for paclitaxel (Taxol) biomimetics. We have successfully used Oregon Green 488 paclitaxel to label microtubules of live HeLa (), NIH 3T3, A-10 and BC3H1 cells. Xenopus laevis and bovine brain microtubules have also been stained with Oregon Green 488 paclitaxel.

In the BODIPY FL and BODIPY 564/570 paclitaxel derivatives, the N-benzoyl substituent of the 3-phenylisoserine portion of native paclitaxel is replaced by a BODIPY propionyl substituent (). As an alternative to chemically modifying tubulin with a reactive fluorophore, a published method describes the use of these BODIPY paclitaxel derivatives to generate fluorescent microtubules that are stable at room temperature for one week or longer. In contrast to the Oregon Green 488 derivative, the BODIPY FL and BODIPY 564/570 paclitaxel derivatives do not appear to be suitable for labeling intracellular tubulin in most cases.

GFP- and RFP-Labeled Tubulin and MAP4

GFP–tubulin fusions are well-established probes for imaging cytokinesis and other dynamic rearrangements of microtubules in live cells. CellLight Tubulin-GFP and CellLight Tubulin-RFP expression vectors (C10613, C10614; CellLight reagents and their targeting sequences—Table 11.1; Figure 11.2.1) generate autofluorescent proteins fused to the N-terminus of human β-tubulin and incorporate all the generic advantages of BacMam 2.0 delivery technology (BacMam Gene Delivery and Expression Technology—Note 11.1).

In context-specific instances where GFP–tubulin fusion protein incorporation into microtubules is inefficient, CellLight expression vectors encoding GFP (C10598) or RFP (C10599) fused to the N-terminus of the mammalian microtubule-associated protein MAP4 provide a second option for microtubule visualization. However, because MAP4 stabilizes polymerized tubulin, CellLight Tubulin-GFP and CellLight Tubulin-RFP are generally preferable for molecular-level investigations of microtubule dynamic instability.

Figure 11.2.1 Human mesenchymal stem cell labeled with CellLight Tubulin-GFP (C10613) and CellLight Histone 2B-RFP (C10595) reagents.

Anti–α-Tubulin Monoclonal Antibody

When used in conjunction with an anti–mouse IgG secondary immunoreagent (Secondary Immunoreagents—Section 7.2, Summary of Molecular Probes secondary antibody conjugates—Table 7.1), our anti–α-tubulin monoclonal antibody (A11126) enables researchers to visualize microtubules in fixed cells (, , , ) and in fixed or frozen tissue sections from various species. This mouse monoclonal antibody, which recognizes amino acid residues 69–97 of the N-terminal structural domain, is also useful for detecting tubulin by ELISA or Western blotting, for screening expression libraries and as a probe for the N-terminal domain of α-tubulin.

The anti–α-tubulin monoclonal antibody is available either unlabeled (A11126) or as a biotin-XX conjugate (A21371). For detecting the biotinylated antibody, we carry a wide variety of fluorophore- and enzyme-labeled avidin, streptavidin and NeutrAvidin biotin-binding protein conjugates and NANOGOLD and Alexa Fluor FluoroNanogold streptavidin (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6, Molecular Probes avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates—Table 7.23).

We have extensively utilized the mouse IgG₁ monoclonal anti–α-tubulin antibody during development and evaluation of our Zenon technology (Zenon Technology: Versatile Reagents for Immunolabeling—Section 7.3, Zenon Antibody Labeling Kits—Table 7.14), which allows labeling of submicrogram quantities of primary antibodies in minutes (, ). A comprehensive listing of our primary antibodies for cytoskeletal proteins can be found at www.invitrogen.com/handbook/antibodies.

BODIPY FL Vinblastine

BODIPY FL vinblastine (V12390, ), a fluorescent analog of the anticancer drug vinblastine, is a useful probe for labeling β-tubulin and for investigating drug-transport mechanisms. Vinblastine inhibits cell proliferation by capping microtubule ends, thereby suppressing mitotic spindle microtubule dynamics. Another fluorescent vinblastine derivative, vinblastine 4'-anthranilate, reportedly binds to the central portion of the primary sequence of β-tubulin and inhibits polymerization.

In addition, intracellular accumulation of vinblastine has been associated with a vinblastine-specific modulating site on P-glycoprotein, a drug-efflux pump that is overexpressed in multidrug-resistant (MDR) cells (Probes for Cell Adhesion, Chemotaxis, Multidrug Resistance and Glutathione—Section 15.6). This highly lipophilic P-glycoprotein substrate has also been used to study the role of P-glycoprotein in drug penetration through the blood-brain barrier. Fluorescently labeled vinblastine analogs, including BODIPY FL vinblastine, have been employed to measure drug-transport kinetics in MDR cells.

Other Probes for Tubulin

The nuclear stain DAPI (D1306, D3571, D21490) binds tightly to purified tubulin in vitro without interfering with microtubule assembly or GTP hydrolysis. DAPI binds to tubulin at sites different from those of paclitaxel, colchicine and vinblastine, and its binding is accompanied by shifts in the absorption spectra and fluorescence enhancement. The affinity of DAPI for polymeric tubulin is sevenfold greater than for dimeric tubulin, making DAPI a sensitive tool for investigating microtubule assembly kinetics. DAPI has been used to screen for potential antimicrotubule drugs in a high-throughput assay.

Bis-ANS (B153) is a potent inhibitor of in vitro microtubule assembly. This fluorescent probe binds to the hydrophobic clefts of proteins with an affinity approximately 10–100 times higher than that of 1,8-ANS (A47, Other Nonpolar and Amphiphilic Probes—Section 13.5) and exhibits a significant fluorescence enhancement upon binding. The bis-ANS binding site on tubulin lies near the critical contact region for microtubule assembly, but it is distinct from the binding sites for colchicine, vinblastine, podophyllotoxin and maytansine. Bis-ANS was used to investigate structural changes in tubulin monomers and dimers during time- and temperature-dependent decay.

DCVJ (4-(dicyanovinyl)julolidine; D3923), which binds to a specific site on the tubulin dimer, has been reported to be a useful probe for following polymerization of tubulin in live cells. DCVJ staining in live cells is mostly blocked by cytochalasin D. Additionally, DCVJ emits strong green fluorescence upon binding to bovine brain calmodulin. The hydrophobic surfaces of tubulin have also been investigated with the environment-sensitive probes nile red (N1142) and prodan (P248).

GFP- and RFP-Labeled Talin

Talin is a cytoskeletal protein that is concentrated in focal adhesions, linking integrins to the actin cytoskeleton either directly or indirectly by interacting with vinculin and α-actinin. CellLight Talin-GFP and CellLight Talin-RFP expression vectors (C10611, C10612; CellLight reagents and their targeting sequences—Table 11.1; Figure 11.2.2) generate autofluorescent proteins fused to the C-terminal actin-binding domain of human talin and incorporate all the generic advantages of BacMam 2.0 delivery technology (BacMam Gene Delivery and Expression Technology—Note 11.1). These CellLight reagents have potential applications in image-based high-content screening (HCS) assays of integrin-mediated cell adhesion, as well as for general-purpose labeling of cytoskeletal actin in live cells.

Figure 11.2.2 HeLa cell labeled with CellLight Talin-GFP (C10611) and Cellular Lights Actin-RFP (C10127) reagents.

Anti–Glial Fibrillary Acidic Protein (GFAP) Antibody

The 50,000-dalton type-III intermediate filament protein known as glial fibrillary acidic protein (GFAP) is a major structural component of astrocytes and some ependymal cells. GFAP associates with the calcium-binding protein annexin II2-p11(2) and S-100. Association with these proteins together with phosphorylation regulates GFAP polymerization. Astrocytes respond to brain injury by proliferation (astrogliosis); one of the first events to occur during astrocyte proliferation is increased GFAP expression. Our anti-GFAP antibody (A21282) and its Alexa Fluor 488 and Alexa Fluor 594 conjugates (A21294, A21295; ) can be used to aid in the identification of cells of glial lineage. Interestingly, antibodies to GFAP have been detected in individuals with dementia. In the central nervous system, anti-GFAP antibody stains both astrocytes and ependymal cells. In the peripheral nervous system, Schwann cells, satellite cells and enteric glial cells are stained; tumors of glial origin contain high amounts of GFAP. No positive staining is observed in skin, connective tissue, adipose tissue, lymphatic tissue, muscle, kidney, ureter, bladder or gastrointestinal tract, including liver and pancreas. Our anti-GFAP antibody does not crossreact with vimentin, which is frequently co-expressed in glioma cells and some astrocytes, nor does it crossreact with Bergmann glia cells, gliomas or other glial cell–derived tumors.

Anti-Desmin Antibody

Desmin, encoded by a gene belonging to the intermediate filament protein gene family, is the main intermediate filament in mature skeletal, cardiac and smooth muscle cells. Both striated and smooth muscle cells can be labeled by an anti-desmin antibody, although not all muscle tissue contains desmin (e.g., aorta smooth muscle). Identification of desmin is useful in distinguishing habdomyosarcomas and leiomyosarcomas from other vimentin-positive sarcomas. We offer a mouse IgG₁ monoclonal anti-desmin antibody (A21283), which can be used with our fluorescent secondary antibodies (Secondary Immunoreagents—Section 7.2, ) as a marker for typing soft tissue sarcomas. Anti-desmin immunohistochemical staining in cell-block preparations may also be helpful in distinguishing mesothelial cells from carcinoma.

Anti-Synapsin I Antibody

Synapsin I, an actin-binding protein, is localized exclusively to synaptic vesicles and thus serves as an excellent marker for synapses in brain and other neuronal tissues. Synapsin I inhibits neurotransmitter release, an effect that is abolished upon its phosphorylation by Ca²⁺/calmodulin–dependent protein kinase II. For assaying the localization and abundance of synapsin I by western blot analysis, immunohistochemistry (), enzyme-linked immunosorbent assay (ELISA) or immunoprecipitation, we offer a polyclonal rabbit anti–synapsin I antibody as an affinity-purified IgG fraction (A6442). Although raised against bovine synapsin I, this antibody also recognizes human, rat and mouse synapsin I; it has little or no activity against synapsin II.