Not all benzimidazole derivatives are microtubule destabilizing agents.

In recent years, microtubule-targeting agents (MTAs) have gained considerable interest in developing novel small-molecule anticancer drugs. MTAs demonstrate anticancer activity either as microtubule-stabilizing agents (paclitaxel) or microtubule-destabilizing agents (nocodazole). FDA-approved drugs containing a benzimidazole ring (nocodazole, albendazole, mebendazole, etc.) are well-known microtubule-destabilizing agents. Thus, most recent research on benzimidazole scaffold-based MTAs focuses on developing microtubule-destabilizing agents. However, there is no report on the benzimidazole scaffold-based microtubule-stabilizing agent. Here, we present the benzimidazole derivatives NI-11 and NI-18 that showed a profound anticancer activity as microtubule-stabilization agents. About twenty benzimidazole analogues were synthesized with excellent yield (80.0% ∼ 98.0%) and tested for their anticancer activity using two cancer cell lines (A549, MCF-7) and one normal cell line (MRC-5). NI-11 showed IC50 values of 2.90, 7.17, and 16.9 µM in A549, MCF-7, and MRC-5 cell lines. NI-18 showed IC50 values of 2.33, 6.10, and 12.1 µM in A549, MCF-7, and MRC-5 cell lines. Thus, NI-11 and NI-18 demonstrated selectivity indexes of 5.81 and 5.20, respectively, which are much higher than the currently available anticancer agents. NI-11 and NI-18 inhibited the cancer cell motility and migration, induced the early phase apoptosis. Both of these comounds were found to show an upregulation of DeY-α-tubulin and downregulation of Ac-α-tubulin expressions in cancer cells. Eventhough the reported benzimidazole scaffold-based commercially available drugs are known to be microtubule-destabilizing agents, the analogues NI-11 and NI-18 were found to have microtubule-stabilizing activity. The in vitro tubulin polymerization assay and the immunofluorescence assay results indicate that the NI-11 and NI-18 exhibit anticancer activity by stabilizing the microtubule network.

Measurement of Microtubule Stability in Mammalian Cells.

The microtubule cytoskeleton is essential for various biological processes such as the intracellular distribution of molecules and organelles, cell morphogenesis, chromosome segregation, and specification of the location of contractile ring formation. Distinct cell types contain microtubules with different extents of stability. For example, microtubules in neurons are highly stabilized to support organelle (or vesicular) transport over large distances, and microtubules in motile cells are more dynamic. In some cases, such as the mitotic spindle, both dynamic and stable microtubules coexist. Alteration of microtubule stability is connected to disease states, making understanding microtubule stability an important area of research. Methods to measure microtubule stability in mammalian cells are described here. Together, these approaches allow microtubule stability to be measured qualitatively or semiquantitatively following staining for post-translational modifications of tubulin or treating cells with microtubule destabilizing agents such as nocodazole. Microtubule stability can also be measured quantitatively by performing fluorescence recovery after photobleaching or fluorescence photoactivation of tubulin in live cells. These methods should be helpful for those seeking to understand microtubule dynamics and stabilization. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Fixing and staining cells for tubulin post-translational modifications Basic Protocol 2: Evaluating microtubule stability following treatment with nocodazole in live or fixed cells Basic Protocol 3: Measurement of microtubule dynamic turnover by quantification of fluorescence recovery after photobleaching Basic Protocol 4: Measurement of microtubule dynamic turnover by quantification of dissipation of fluorescence after photoactivation.

HUSH-mediated HIV silencing is independent of TASOR phosphorylation on threonine 819.

BACKGROUND: TASOR, a component of the HUSH repressor epigenetic complex, and SAMHD1, a cellular triphosphohydrolase (dNTPase), are both anti-HIV proteins antagonized by HIV-2/SIVsmm Viral protein X. As a result, the same viral protein is able to relieve two different blocks along the viral life cell cycle, one at the level of reverse transcription, by degrading SAMHD1, the other one at the level of proviral expression, by degrading TASOR. Phosphorylation of SAMHD1 at T592 has been shown to downregulate its antiviral activity. The discovery that T819 in TASOR was lying within a SAMHD1 T592-like motif led us to ask whether TASOR is phosphorylated on this residue and whether this post-translational modification could regulate its repressive activity. RESULTS: Using a specific anti-phospho-antibody, we found that TASOR is phosphorylated at T819, especially in cells arrested in early mitosis by nocodazole. We provide evidence that the phosphorylation is conducted by a Cyclin/CDK1 complex, like that of SAMHD1 at T592. While we could not detect TASOR in quiescent CD4 + T cells, TASOR and its phosphorylated form are present in activated primary CD4 + T lymphocytes. In addition, TASOR phosphorylation appears to be independent from TASOR repressive activity. Indeed, on the one hand, nocodazole barely reactivates HIV-1 in the J-Lat A1 HIV-1 latency model despite TASOR T819 phosphorylation. On the other hand, etoposide, a second cell cycle arresting drug, reactivates latent HIV-1, without concomitant TASOR phosphorylation. Furthermore, overexpression of wt TASOR or T819A or T819E similarly represses gene expression driven by an HIV-1-derived LTR promoter. Finally, while TASOR is degraded by HIV-2 Vpx, TASOR phosphorylation is prevented by HIV-1 Vpr, likely as a consequence of HIV-1 Vpr-mediated-G2 arrest. CONCLUSIONS: Altogether, we show that TASOR phosphorylation occurs in vivo on T819. This event does not appear to correlate with TASOR-mediated HIV-1 silencing. We speculate that TASOR phosphorylation is related to a role of TASOR during cell cycle progression.

Map7D2 and Map7D1 facilitate microtubule stabilization through distinct mechanisms in neuronal cells.

Microtubule (MT) dynamics are modulated through the coordinated action of various MT-associated proteins (MAPs). However, the regulatory mechanisms underlying MT dynamics remain unclear. We show that the MAP7 family protein Map7D2 stabilizes MTs to control cell motility and neurite outgrowth. Map7D2 directly bound to MTs through its N-terminal half and stabilized MTs in vitro. Map7D2 localized prominently to the centrosome and partially on MTs in mouse N1-E115 neuronal cells, which expresses two of the four MAP7 family members, Map7D2 and Map7D1. Map7D2 loss decreased the resistance to the MT-destabilizing agent nocodazole without affecting acetylated/detyrosinated stable MTs, suggesting that Map7D2 stabilizes MTs via direct binding. In addition, Map7D2 loss increased the rate of random cell migration and neurite outgrowth, presumably by disturbing the balance between MT stabilization and destabilization. Map7D1 exhibited similar subcellular localization and gene knockdown phenotypes to Map7D2. However, in contrast to Map7D2, Map7D1 was required for the maintenance of acetylated stable MTs. Taken together, our data suggest that Map7D2 and Map7D1 facilitate MT stabilization through distinct mechanisms in cell motility and neurite outgrowth.

Rapid profiling of G2 phase to mitosis progression by flow cytometry in asynchronous cells.

The precise control of the cell cycle G2 phase to Mitosis (M phase) transition is central for cell fate determination. The commonly used methods for assessing G2 to M phase progression are based on synchronizing cells and involve perturbation of the natural cell cycle progression. Additionally, these methods are often time-consuming and labor-intensive. Here, we report a flow cytometry-based method that offers a kinetic analysis of G2 to M phase progression in asynchronous cells using nocodazole, 5-Ethynyl-2´-deoxyuridine staining, and histone H3 serine 28 phosphorylation (pH3) staining. Nocodazole is used to collect mitotic cells and prevent their progression into G1, at the same time EdU is added for use as a dump channel during analysis. The remaining cells can then be identified as either G1 or G2/M based on their DNA content. Finally, G2 and M phase cells can be separated based on a mitotic marker, phosphorylation of ser28 on histone H3. While developed to assay G2/M phase progression, this method also resolves G1/S phase progression with no additional steps other than analysis. Compared to double thymidine block, this method does not require extended pre-treatments and is compatible with a greater variety of cell lines, while at the same time offering enhanced consistency and temporal resolution.

RNA-sequencing Profiles of Cell Cycle-Related Genes Upregulated during the G2-Phase in Giardia lamblia.

To identify the component(s) involved in cell cycle control in the protozoan Giardia lamblia, cells arrested at the G1/S- or G2-phase by treatment with nocodazole and aphidicolin were prepared from the synchronized cell cultures. RNA-sequencing analysis of the 2 stages of Giardia cell cycle identified several cell cycle genes that were up-regulated at the G2-phase. Transcriptome analysis of cells in 2 distinct cell cycle stages of G. lamblia confirmed previously reported components of cell cycle (PcnA, cyclin B, and CDK) and identified additional cell cycle components (NEKs, Mad2, spindle pole protein, and CDC14A). This result indicates that the cell cycle machinery operates in this protozoan, one of the earliest diverging eukaryotic lineages.

Dynamic Microtubules in Alzheimer's Disease: Association with Dendritic Spine Pathology.

Alzheimer's disease (AD) is the most common incurable neurodegenerative disorder that affects the processes of memory formation and storage. The loss of dendritic spines and alteration in their morphology in AD correlate with the extent of patient's cognitive decline. Tubulin had been believed to be restricted to dendritic shafts, until recent studies demonstrated that dynamically growing tubulin microtubules enter dendritic spines and promote their maturation. Abnormalities of tubulin cytoskeleton may contribute to the process of dendritic spine shape alteration and their subsequent loss in AD. In this review, association between tubulin cytoskeleton dynamics and dendritic spine morphology is discussed in the context of dendritic spine alterations in AD. Potential implications of these findings for the development of AD therapy are proposed.

Precocious cleavage furrows simultaneously move and ingress when kinetochore microtubules are depolymerized in Mesostoma ehrenbergii spermatocytes.

A "precocious" cleavage furrow develops and ingresses during early prometaphase in Mesostoma ehrenbergii spermatocytes (Forer and Pickett-Heaps Eur J Cell Biol 89:607-618, 2010). In response to chromosome movements which regularly occur during prometaphase and that alter the balance of chromosomes in the two half-spindles, the precocious furrow shifts its position along the cell, moving 2-3 µm towards the half cell with fewer chromosomes (Ferraro-Gideon et al. Cell Biol Int 37:892-898, 2013). This process continues until proper segregation is achieved and the cell enters anaphase with the cleavage furrow again in the middle of the cell. At anaphase, the furrow recommences ingression. Spindle microtubules (MTs) are implicated in various furrow positioning models, and our experiments studied the responses of the precocious furrows to the absence of spindle MTs. We depolymerized spindle MTs during prometaphase using various concentrations of nocodazole (NOC) and colcemid. The expected result is that the furrow should regress and chromosomes remain in the midzone of the cell (Cassimeris et al. J Cell Sci 96:9-15, 1990). Instead, the furrows commenced ingression and all three bivalent chromosomes moved to one pole while the univalent chromosomes, that usually reside at the two poles, either remained at their poles or moved to the opposite pole along with the bivalents, as described elsewhere (Fegaras and Forer 2018). The microtubules were completely depolymerized by the drugs, as indicated by immunofluorescence staining of treated cells (Fegaras and Forer 2018), and in the absence of microtubules, the furrows often ingressed (in 33/61 cells) at a rate similar to normal anaphase ingression (~ 1 µm/min), while often simultaneously moving toward one pole. Thus, these results indicate that in the absence of anaphase and of spindle microtubules, cleavage furrows resume ingression.

Effects of the Protonation State of Titratable Residues and the Presence of Water Molecules on Nocodazole Binding to ß-Tubulin.

Regulation of microtubule assembly by antimitotic agents is a potential therapeutic strategy for the treatment of cancer, parasite infections, and neurodegenerative diseases. One of these agents is nocodazole (NZ), which inhibits microtubule polymerization by binding to ß-tubulin. NZ was recently co-crystallized in Gallus gallus tubulin, providing new information about the features of interaction for ligand recognition and stability. In this work, we used state-of-the-art computational approaches to evaluate the protonation effects of titratable residues and the presence of water molecules in the binding of NZ. Analysis of protonation states showed that residue E198 has the largest modification in its pKa value. The resulting E198 pKa value, calculated with pH-REMD methodology (pKa =6.21), was higher than the isolated E amino acid (pKa =4.25), thus being more likely to be found in its protonated state at the binding site. Moreover, we identified an interaction between a water molecule and C239 and G235 as essential for NZ binding. Our results suggest that the protonation state of E198 and the structural water molecules play key roles in the binding of NZ to ß-tubulin.

Leukaemic cells from chronic lymphocytic leukaemia patients undergo apoptosis following microtubule depolymerization and Lyn inhibition by nocodazole.

Functional abnormalities of chronic lymphocytic leukaemia (CLL) cells may be related to the microtubular network of cell cytoskeleton; specifically tubulin involvement in cells after B-cell receptor engagement. As microtubule inhibitors could represent a therapeutic strategy for CLL, this study investigated the capability of nocodazole, a synthetic depolymerizing agent, to kill CLL leukaemic cells. We demonstrated that nocodazole was highly specific for the in vitro induction of apoptosis in leukaemic cells from 90 CLL patients, without affecting the viability of T-cells and/or mesenchymal stromal cells (MSCs) recovered from the same patients. Nocodazole was observed to overcome the pro-survival signals provided by MSCs. Competing with ATP for the nucleotide-binding site, nocodazole has been observed to turn off the high basal tyrosine phosphorylation of leukaemic cells mediated by the Src-kinase Lyn. Considering that most anti-microtubule drugs have limited clinical use because of their strong toxic effects, the high selectivity of nocodazole for leukaemic cells in CLL and its capability to bypass microenvironmental pro-survival stimuli, suggests the use of this inhibitor for designing new therapeutic strategies in CLL treatment.

Discovery of new benzothiazole-based inhibitors of breakpoint cluster region-Abelson kinase including the T315I mutant.

The existence of drug resistance caused by mutations in the break-point cluster region-Abelson tyrosine kinase (Bcr-Abl) kinase domain remains a clinical challenge due to limited effective treatment options for chronic myeloid leukemia (CML). Herein we report a novel series of benzothiazole-based inhibitors that are effective against wild-type and T315I mutant Bcr-Abl kinases. The original hit compound, nocodazole, was extensively modified through a structure-based drug design strategy, especially by varying the groups at the C2 and C6 positions of the scaffold. In addition, the introduction of water-solubilizing groups at the terminal ethyl group resulted in enhanced physicochemical properties and potency in cellular inhibition. Several compounds inhibited the kinase activity of both wild-type Bcr-Abl and the T315I mutant with IC50 values in the picomolar range and exhibited good antiproliferative effects on Ba/F3 cell lines transformed with either wild-type or T315I mutant Bcr-Abl.

Acoustic detection of melanosome transport in Xenopus laevis melanophores.

Organelle transport studies are often performed using melanophores from lower vertebrates due to the ease of inducing movements of pigment granules (melanosomes) and visualizing them by optical microscopy. Here, we present a novel methodology to monitor melanosome translocation (which is a light-sensitive process) in the dark using the quartz crystal microbalance with dissipation monitoring (QCM-D) technique. This acoustic sensing method was used to study dispersion and aggregation of melanosomes in Xenopus laevis melanophores. Reversible sensor responses, correlated to optical reflectance measurements, were obtained by alternating addition and removal of melatonin (leading to melanosome aggregation) and melanocyte-stimulating hormone (MSH) (leading to melanosome dispersion). By confocal microscopy, it was shown that a vertical redistribution of melanosomes occurred during the dispersion/aggregation processes. Furthermore, the transport process was studied in the presence of cytoskeleton-perturbing agents disrupting either actin filaments (latrunculin) or microtubules (nocodazole). Taken together, these experiments suggest that the acoustic responses mainly originate from melanosome transport along actin filaments (located close to the cell membrane), as expected based on the penetration depth of the QCM-D technique. The results clearly indicate the potential of QCM-D for studies of intracellular transport processes in melanophores.

Prolonged prometaphase blocks daughter cell proliferation despite normal completion of mitosis.

The mitotic checkpoint maintains genomic stability by blocking the metaphase-anaphase transition until all kinetochores attach to spindle microtubules [1, 2]. However, some defects are not detected by this checkpoint. With low concentrations of microtubule-targeting agents, the checkpoint eventually becomes satisfied, though the spindles may be short and/or multipolar [3, 4] and the fidelity of chromosome distribution and cleavage completion are compromised. In real life, environmental toxins, radiation, or chemotherapeutic agents may lead to completed but inaccurate mitoses. It has been assumed that once the checkpoint is satisfied and cells divide, the daughter cells would proliferate regardless of prometaphase duration. However, when continuously exposed to microtubule inhibitors, untransformed cells eventually slip out of mitosis after 12-48 hr and arrest in G1 [5-8] (see also [9]). Interestingly, transient but prolonged treatments with nocodazole allow completion of mitosis, but the daughter cells arrest in interphase [10, 11] (see also [9, 12]). Here we characterize the relationship between prometaphase duration and the proliferative capacity of daughter cells. Our results reveal the existence of a mechanism that senses prometaphase duration; if prometaphase lasts >1.5 hr, this mechanism triggers a durable p38- and p53-dependent G1 arrest of the daughter cells despite normal division of their mothers.

Dishevelled, a Wnt signalling component, is involved in mitotic progression in cooperation with Plk1.

Wnt signalling is known to promote G1/S progression through the stimulation of gene expression, but whether this signalling regulates mitotic progression is not clear. Here, the function of dishevelled 2 (Dvl2), which transmits the Wnt signal, in mitosis was examined. Dvl2 localized to the spindles and spindle poles during mitosis. When cells were treated with nocodazole, Dvl2 was observed at the kinetochores (KTs). Dvl2 bound to and was phosphorylated at Thr206 by a mitotic kinase, Polo-like kinase 1 (Plk1), and this phosphorylation was required for spindle orientation and stable microtubule (MT)-KT attachment. Dvl2 was also found to be involved in the activation of a spindle assembly checkpoint (SAC) kinase, Mps1, and the recruitment of other SAC components, Bub1 and BubR1, to the KTs. However, the phosphorylation of Dvl2 by Plk1 was dispensable for SAC. Furthermore, Wnt receptors were involved in spindle orientation, but not in MT-KT attachment or SAC. These results suggested that Dvl2 is involved in mitotic progression by regulating the dynamics of MT plus-ends and the SAC in Plk1-dependent and -independent manners.

The APC/C recruits cyclin B1-Cdk1-Cks in prometaphase before D box recognition to control mitotic exit.

The ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C) is activated at prometaphase by mitotic phosphorylation and binding of its activator, Cdc20. This initiates cyclin A degradation, whereas cyclin B1 is stabilized by the spindle checkpoint. Upon checkpoint release, the RXXL destruction box (D box) was proposed to direct cyclin B1 to core APC/C or Cdc20. In this study, we report that endogenous cyclin B1-Cdk1 is recruited to checkpoint-inhibited, phosphorylated APC/C in prometaphase independently of Cdc20 or the cyclin B1 D box. Like cyclin A, cyclin B1 binds the APC/C by the Cdk cofactor Cks and the APC3 subunit. Prior binding to APC/C(Cdc20) makes cyclin B1 a better APC/C substrate in metaphase, driving mitotic exit and cytokinesis. We conclude that in prometaphase, the phosphorylated APC/C can recruit both cyclin A and cyclin B1 in a Cks-dependent manner. This suggests that the spindle checkpoint blocks D box recognition of APC/C-bound cyclin B1, whereas distinctive complexes between the N terminus of cyclin A and Cdc20 evade checkpoint control.

The cytoskeletal organization of breast carcinoma and fibroblast cells inside three dimensional (3-D) isotropic silicon microstructures.

Studying the cytoskeletal organization as cells interact in their local microenvironment is interest of biological science, tissue engineering and cancer diagnosis applications. Herein, we describe the behavior of cell lines obtained from metastatic breast tumor pleural effusions (MDA-MB-231), normal fibrocystic mammary epithelium (MCF10A), and HS68 normal fibroblasts inside three dimensional (3-D) isotropic silicon microstructures fabricated by a single-mask, single-isotropic-etch process. We report differences in adhesion, mechanism of force balance within the cytoskeleton, and deformability among these cell types inside the 3-D microenvironment. HS68 fibroblasts typically stretched and formed vinculin-rich focal adhesions at anchor sites inside the etched cavities. In contrast, MCF10A and MDA-MB-231 cells adopted the curved surfaces of isotropic microstructures and exhibited more diffuse vinculin cytoplasmic staining in addition to vinculin localized in focal adhesions. The measurement of cells elasticity using atomic force microscopy (AFM) indentation revealed that HS68 cells are significantly stiffer (p < 0.0001) than MCF10A and MDA-MB-231 cells. Upon microtubule disruption with nocodazole, fibroblasts no longer stretched, but adhesion of MCF10A and MDA-MB-231 within the etched features remained unaltered. Our findings are consistent with tensegrity theory. The 3-D microstructures have the potential to probe cytoskeletal-based differences between healthy and diseased cells that can provide biomarkers for diagnostics purposes.

Identification and characterisation of new inhibitors for the human hematopoietic prostaglandin D2 synthase.

Prostaglandin D(2) synthesised by the hematopoietic prostaglandin D(2) synthase has a pro-inflammatory effect in allergic asthma, regulating many hallmark characteristics of the disease. Here we describe identification of hematopoietic prostaglandin D(2) synthase inhibitors including cibacron blue, bromosulfophthalein and ethacrynic acid. Expansion around the drug-like ethacrynic acid identified a novel inhibitor, nocodazole, and a fragment representing its aromatic core. Nocodazole binding was further characterised by docking calculations in combination with conformational strain analysis. The benzyl thiophene core was predicted to be buried in the active site, binding in the putative prostaglandin binding site, and a likely hydrogen bond donor site identified. X-ray crystallographic studies supported the predicted binding mode.

Brd4 marks select genes on mitotic chromatin and directs postmitotic transcription.

On entry into mitosis, many transcription factors dissociate from chromatin, resulting in global transcriptional shutdown. During mitosis, some genes are marked to ensure the inheritance of their expression in the next generation of cells. The nature of mitotic gene marking, however, has been obscure. Brd4 is a double bromodomain protein that localizes to chromosomes during mitosis and is implicated in holding mitotic memory. In interphase, Brd4 interacts with P-TEFb and functions as a global transcriptional coactivator. We found that throughout mitosis, Brd4 remained bound to the transcription start sites of many M/G1 genes that are programmed to be expressed at the end of, or immediately after mitosis. In contrast, Brd4 did not bind to genes that are expressed at later phases of cell cycle. Brd4 binding to M/G1 genes increased at telophase, the end phase of mitosis, coinciding with increased acetylation of histone H3 and H4 in these genes. Increased Brd4 binding was accompanied by the recruitment of P-TEFb and de novo M/G1 gene transcription, the events impaired in Brd4 knockdown cells. In sum, Brd4 marks M/G1 genes for transcriptional memory during mitosis, and upon exiting mitosis, this mark acts as a signal for initiating their prompt transcription in daughter cells.

Motor-dependent microtubule disassembly driven by tubulin tyrosination.

In cells, stable microtubules (MTs) are covalently modified by a carboxypeptidase, which removes the C-terminal Tyr residue of alpha-tubulin. The significance of this selective detyrosination of MTs is not understood. In this study, we report that tubulin detyrosination in fibroblasts inhibits MT disassembly. This inhibition is relieved by overexpression of the depolymerizing motor mitotic centromere-associated kinesin (MCAK). Conversely, suppression of MCAK expression prevents disassembly of normal tyrosinated MTs in fibroblasts. Detyrosination of MTs suppresses the activity of MCAK in vitro, apparently as the result of a decreased affinity of the adenosine diphosphate (ADP)-inorganic phosphate- and ADP-bound forms of MCAK for the MT lattice. Detyrosination also impairs MT disassembly in neurons and inhibits the activity of the neuronal depolymerizing motor KIF2A in vitro. These results indicate that MT depolymerizing motors are directly inhibited by the detyrosination of tubulin, resulting in the stabilization of cellular MTs. Detyrosination of transiently stabilized MTs may give rise to persistent subpopulations of disassembly-resistant polymers to sustain subcellular cytoskeletal differentiation.