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Mitotic centromere-associated kinesin (MCAK) motor protein is a typical member of the kinesin-13 family, which can depolymerize microtubules from both plus and minus ends. A critical issue for the MCAK motor is how it performs the depolymerase activity. To address the issue, the pathway of the MCAK motor moving on microtubules and depolymerizing the microtubules is presented here. On the basis of the pathway, the dynamics of both the wild-type and mutant MCAK motors is studied theoretically, which include the full-length MCAK, the full-length MCAK with mutations in the α4-helix of the motor domain, the mutant full-length MCAK with a neutralized neck, the monomeric MCAK and the mutant monomeric MCAK with a neutralized neck. The studies show that a single dimeric MCAK motor can depolymerize microtubules in a processive manner, with either one tubulin or two tubulins being removed per times. The theoretical results are in agreement with the available experimental data. Moreover, predicted results are provided. [ABSTRACT FROM AUTHOR]

Salvinal is a natural lignan isolated from the roots of Salvia mitorrhiza Bunge (Danshen). Previous studies have demonstrated its anti-proliferative activity in both drug-sensitive and -resistant cancer cell lines, with IC50 values ranging from 4–17 µM. In this study, a series of salvinal derivatives was synthesized and evaluated for the structure–activity relationship. Among the twenty-four salvinal derivatives, six compounds showed better anticancer activity than salvinal. Compound 25 displayed excellent anticancer activity, with IC50 values of 0.13–0.14 µM against KB, KB-Vin10 (overexpress MDR/Pgp), and KB-7D (overexpress MRP) human carcinoma cell lines. Based on our in vitro microtubule depolymerization assay, compound 25 showed depolymerization activity in a dose-dependent manner. Our findings indicate that compound 25 is a promising anticancer agent with depolymerization activity that has potential for the management of malignance. [ABSTRACT FROM AUTHOR]

The pivotal role of cysteine-rich receptor-like kinases (CRKs) in modulating growth, development, and responses to stress has been widely acknowledged in Arabidopsis. However, the function and regulation of CRK41 has remained unclear. In this study, we demonstrate that CRK41 is critical for modulating microtubule depolymerization in response to salt stress. The crk41 mutant exhibited increased tolerance, while overexpression of CRK41 led to hypersensitivity to salt. Further analysis revealed that CRK41 interacts directly with the MAP kinase3 (MPK3), but not with MPK6. Inactivation of either MPK3 or MPK6 could abrogate the salt tolerance of the crk41 mutant. Upon NaCl treatment, microtubule depolymerization was heightened in the crk41 mutant, yet alleviated in the crk41mpk3 and crk41mpk6 double mutants, indicating that CRK41 suppresses MAPK-mediated microtubule depolymerizations. Collectively, these results reveal that CRK41 plays a crucial role in regulating microtubule depolymerization triggered by salt stress through coordination with MPK3/MPK6 signalling pathways, which are key factors in maintaining microtubule stability and conferring salt stress resistance in plants. [ABSTRACT FROM AUTHOR]

Centrosome-containing cells assemble their spindles exploiting three main classes of microtubules (MTs): MTs nucleated by the centrosomes, MTs generated near the chromosomes/kinetochores, and MTs nucleated within the spindle by the augmin-dependent pathway. Mammalian and Drosophila cells lacking the centrosomes generate MTs at kinetochores and eventually form functional bipolar spindles. However, the mechanisms underlying kinetochore-driven MT formation are poorly understood. One of the ways to elucidate these mechanisms is the analysis of spindle reassembly following MT depolymerization. Here, we used an RNA interference (RNAi)-based reverse genetics approach to dissect the process of kinetochore-driven MT regrowth (KDMTR) after colcemid-induced MT depolymerization. This MT depolymerization procedure allows a clear assessment of KDMTR, as colcemid disrupts centrosome-driven MT regrowth but not KDMTR. We examined KDMTR in normal Drosophila S2 cells and in S2 cells subjected to RNAi against conserved genes involved in mitotic spindle assembly: mast/orbit/chb (CLASP1), mei-38 (TPX2), mars (HURP), dgt6 (HAUS6), Eb1 (MAPRE1/EB1), Patronin (CAMSAP2), asp (ASPM), and Klp10A (KIF2A). RNAi-mediated depletion of Mast/Orbit, Mei-38, Mars, Dgt6, and Eb1 caused a significant delay in KDMTR, while loss of Patronin had a milder negative effect on this process. In contrast, Asp or Klp10A deficiency increased the rate of KDMTR. These results coupled with the analysis of GFP-tagged proteins (Mast/Orbit, Mei-38, Mars, Eb1, Patronin, and Asp) localization during KDMTR suggested a model for kinetochore-dependent spindle reassembly. We propose that kinetochores capture the plus ends of MTs nucleated in their vicinity and that these MTs elongate at kinetochores through the action of Mast/Orbit. The Asp protein binds the MT minus ends since the beginning of KDMTR, preventing excessive and disorganized MT regrowth. Mei-38, Mars, Dgt6, Eb1, and Patronin positively regulate polymerization, bundling, and stabilization of regrowing MTs until a bipolar spindle is reformed. [ABSTRACT FROM AUTHOR]

Drought stress is a common abiotic challenge that profoundly impacts plant growth and development. As sessile organisms, plants rely on various physiological and morphological adaptations to cope with drought conditions. The CIPK (calcineurin B-like protein-interacting protein kinase) family proteins play a pivotal role in mediating plant responses to abiotic stress through modulation of cellular membrane events via the CBL-CIPK complex. However, reports documenting the CIPKs' regulation of non-membrane events are scant. In this study, we discovered a novel subcellular localisation pattern of the AtCIPK20 protein of Arabidopsis, specifically to cortical microtubules (cMT), which is distinct from previously reported localisation patterns of plant CIPKs. AtCIPK20 regulates ABA-induced loss of cMT organisation in guard cells, thereby facilitating stomatal closure, mitigating leaf water loss, and protecting plants from drought stress in Arabidopsis. The C-terminal regulatory domain of AtCIPK20 governs its cMT targeting, whereas the interaction of AtCIPK20 with its CBL partners disrupts this localisation. Notably, the cMT targeting characteristic of AtCIPK20 is not exclusive, as several other CIPK members in Arabidopsis, maize, and rice exhibit similar localisation patterns. These findings broaden our current understanding of the role of plant CIPK members in abiotic stress resistance and suggest that future exploration of CIPK molecular functions should adopt a more comprehensive perspective., (© 2024 John Wiley & Sons Ltd.)

The activity of a kinesin is largely determined by the approximately 350 residue motor domain, and this region alone is sufficient to classify a kinesin as a member of a particular family. The kinesin-13 family are a group of microtubule depolymerizing kinesins and are vital regulators of microtubule length. Kinesin-13s are critical to spindle assembly and chromosome segregation in both mitotic and meiotic cell division and play crucial roles in cilium length control and neuronal development. To better understand the evolution of microtubule depolymerization activity, we created a synthetic ancestral kinesin-13 motor domain. This phylogenetically inferred ancestral motor domain is the sequence predicted to have existed in the common ancestor of the kinesin-13 family. Here we show that the ancestral kinesin-13 motor depolymerizes stabilized microtubules faster than any previously tested depolymerase. This potent activity is more than an order of magnitude faster than the most highly studied kinesin-13, MCAK and allows the ancestral kinesin-13 to depolymerize doubly stabilized microtubules and cause internal breaks within microtubules. These data suggest that the ancestor of the kinesin-13 family was a ‘super depolymerizer’ and that members of the kinesin-13 family have evolved away from this extreme depolymerizing activity to provide more controlled microtubule depolymerization activity in extant cells.

Diclofenac, a nonsteroidal anti-inflammatory drug (NSAID) used to treat inflammatory diseases induces cellular toxicity by increasing the production of reactive oxygen species (ROS) and impairing autophagic flux. In this study, we investigated whether diclofenac induces cancer cell death and the mechanism by which diclofenac causes cell death. We observed that diclofenac induces mitotic arrest with a half-maximal effective concentration of 170 μM and cell death with a half-maximal lethal dose of 200 µM during 18-h incubation in HeLa cells. Cellular microtubule imaging and in vitro tubulin polymerization assays demonstrated that treatment with diclofenac elicits microtubule destabilization. Autophagy relies on microtubule-mediated transport and the fusion of autophagic vesicles. We observed that diclofenac inhibits both phagophore movement, an early step of autophagy, and the fusion of autophagosomes and lysosomes, a late step of autophagy. Diclofenac also induces the fragmentation of mitochondria and the Golgi during cell death. We found that diclofenac induces cell death further in combination with 5-fuorouracil, a DNA replication inhibitor than in single treatment in cancer cells. Pancreatic cancer cells, which have high basal autophagy, are particularly sensitive to cell death by diclofenac. Our study suggests that microtubule destabilization by diclofenac induces cancer cell death via compromised spindle assembly checkpoints and increased ROS through impaired autophagy flux. Diclofenac may be a candidate therapeutic drug in certain type of cancers by inhibiting microtubule-mediated cellular events in combination with clinically utilized nucleoside metabolic inhibitors, including 5-fluorouracil, to block cancer cell proliferation. [ABSTRACT FROM AUTHOR]

Salvinal is a natural lignan isolated from the roots of Salvia mitorrhiza Bunge (Danshen). Previous studies have demonstrated its anti-proliferative activity in both drug-sensitive and -resistant cancer cell lines, with IC50 values ranging from 4–17 µM. In this study, a series of salvinal derivatives was synthesized and evaluated for the structure–activity relationship. Among the twenty-four salvinal derivatives, six compounds showed better anticancer activity than salvinal. Compound 25 displayed excellent anticancer activity, with IC50 values of 0.13–0.14 µM against KB, KB-Vin10 (overexpress MDR/Pgp), and KB-7D (overexpress MRP) human carcinoma cell lines. Based on our in vitro microtubule depolymerization assay, compound 25 showed depolymerization activity in a dose-dependent manner. Our findings indicate that compound 25 is a promising anticancer agent with depolymerization activity that has potential for the management of malignance.

The pivotal role of cysteine-rich receptor-like kinases (CRKs) in modulating growth, development, and responses to stress has been widely acknowledged in Arabidopsis. However, the function and regulation of CRK41 has remained unclear. In this study, we demonstrate that CRK41 is critical for modulating microtubule depolymerization in response to salt stress. The crk41 mutant exhibited increased tolerance, while overexpression of CRK41 led to hypersensitivity to salt. Further analysis revealed that CRK41 interacts directly with the MAP kinase3 (MPK3), but not with MPK6. Inactivation of either MPK3 or MPK6 could abrogate the salt tolerance of the crk41 mutant. Upon NaCl treatment, microtubule depolymerization was heightened in the crk41 mutant, yet alleviated in the crk41mpk3 and crk41mpk6 double mutants, indicating that CRK41 suppresses MAPK-mediated microtubule depolymerizations. Collectively, these results reveal that CRK41 plays a crucial role in regulating microtubule depolymerization triggered by salt stress through coordination with MPK3/MPK6 signalling pathways, which are key factors in maintaining microtubule stability and conferring salt stress resistance in plants.

Dynamic microtubules play a critical role in cell structure and function. In nervous system, microtubules are the major route for cargo protein trafficking and they specially extend into and out of synapses to regulate synaptic development and plasticity. However, the detailed depolymerization mechanism that regulates dynamic microtubules in synapses and dendrites is still unclear. In this study, we find that KIF2C, a dynamic microtubule depolymerization protein without known function in the nervous system, plays a pivotal role in the structural and functional plasticity of synapses and regulates cognitive function in mice. Through its microtubule depolymerization capability, KIF2C regulates microtubule dynamics in dendrites, and regulates microtubule invasion of spines in neurons in a neuronal activity-dependent manner. Using RNAi knockdown and conditional knockout approaches, we showed that KIF2C regulates spine morphology and synaptic membrane expression of AMPA receptors. Moreover, KIF2C deficiency leads to impaired excitatory transmission, long-term potentiation, and altered cognitive behaviors in mice. Collectively, our study explores a novel function of KIF2C in the nervous system and provides an important regulatory mechanism on how activity-dependent microtubule dynamic regulates synaptic plasticity and cognition behaviors.

Centrosome-containing cells assemble their spindles exploiting three main classes of microtubules (MTs): MTs nucleated by the centrosomes, MTs generated near the chromosomes/kinetochores, and MTs nucleated within the spindle by the augmin-dependent pathway. Mammalian and Drosophila cells lacking the centrosomes generate MTs at kinetochores and eventually form functional bipolar spindles. However, the mechanisms underlying kinetochore-driven MT formation are poorly understood. One of the ways to elucidate these mechanisms is the analysis of spindle reassembly following MT depolymerization. Here, we used an RNA interference (RNAi)-based reverse genetics approach to dissect the process of kinetochore-driven MT regrowth (KDMTR) after colcemid-induced MT depolymerization. This MT depolymerization procedure allows a clear assessment of KDMTR, as colcemid disrupts centrosome-driven MT regrowth but not KDMTR. We examined KDMTR in normal Drosophila S2 cells and in S2 cells subjected to RNAi against conserved genes involved in mitotic spindle assembly: mast/orbit/chb (CLASP1), mei-38 (TPX2), mars (HURP), dgt6 (HAUS6), Eb1 (MAPRE1/EB1), Patronin (CAMSAP2), asp (ASPM), and Klp10A (KIF2A). RNAi-mediated depletion of Mast/Orbit, Mei-38, Mars, Dgt6, and Eb1 caused a significant delay in KDMTR, while loss of Patronin had a milder negative effect on this process. In contrast, Asp or Klp10A deficiency increased the rate of KDMTR. These results coupled with the analysis of GFP-tagged proteins (Mast/Orbit, Mei-38, Mars, Eb1, Patronin, and Asp) localization during KDMTR suggested a model for kinetochore-dependent spindle reassembly. We propose that kinetochores capture the plus ends of MTs nucleated in their vicinity and that these MTs elongate at kinetochores through the action of Mast/Orbit. The Asp protein binds the MT minus ends since the beginning of KDMTR, preventing excessive and disorganized MT regrowth. Mei-38, Mars, Dgt6, Eb1, and Patronin positively regulate polymerization, bundling, and stabilization of regrowing MTs until a bipolar spindle is reformed.

Abstract Background A previous screen of a human kinase and phosphatase shRNA library to select genes that mediate arsenite induction of spindle abnormalities resulted in the identification of phosphatidylinositol-5-phosphate 4-kinase type-2 gamma (PIP4KIIγ), a phosphatidylinositol 4,5-bisphosphate (PIP2)-synthesizing enzyme. In this study, we explored how PIP4KIIγ regulates the assembly of mitotic spindles. Results PIP4KIIγ accumulates at the spindle pole before anaphase, and is required for the assembly of functional bipolar spindles. Depletion of PIP4KIIγ enhanced the spindle pole accumulation of mitotic centromere-associated kinesin (MCAK), a microtubule (MT)-depolymerizing kinesin, and resulted in a less stable spindle pole-associated MT. Depletion of MCAK can ameliorate PIP4KIIγ depletion-induced spindle abnormalities. In addition, PIP2 binds to polo-like kinase (PLK1) and reduces PLK1-mediated phosphorylation of MCAK. These results indicate that PIP4KIIγ and PIP2 may negatively regulate the MT depolymerization activity of MCAK by reducing PLK1-mediated phosphorylation of MCAK. Consequently, depletion of PLK1 has been shown to counteract the PIP4KIIγ depletion-induced instability of spindle pole-associated MT and cell resistance to arsenite. Conclusions Our current results imply that PIP4KIIγ may restrain MT depolymerization at the spindle pole through attenuating PLK1-mediated activation of MCAK before anaphase onset.

Diclofenac, a nonsteroidal anti-inflammatory drug (NSAID) used to treat inflammatory diseases induces cellular toxicity by increasing the production of reactive oxygen species (ROS) and impairing autophagic flux. In this study, we investigated whether diclofenac induces cancer cell death and the mechanism by which diclofenac causes cell death. We observed that diclofenac induces mitotic arrest with a half-maximal effective concentration of 170 μM and cell death with a half-maximal lethal dose of 200 µM during 18-h incubation in HeLa cells. Cellular microtubule imaging and in vitro tubulin polymerization assays demonstrated that treatment with diclofenac elicits microtubule destabilization. Autophagy relies on microtubule-mediated transport and the fusion of autophagic vesicles. We observed that diclofenac inhibits both phagophore movement, an early step of autophagy, and the fusion of autophagosomes and lysosomes, a late step of autophagy. Diclofenac also induces the fragmentation of mitochondria and the Golgi during cell death. We found that diclofenac induces cell death further in combination with 5-fuorouracil, a DNA replication inhibitor than in single treatment in cancer cells. Pancreatic cancer cells, which have high basal autophagy, are particularly sensitive to cell death by diclofenac. Our study suggests that microtubule destabilization by diclofenac induces cancer cell death via compromised spindle assembly checkpoints and increased ROS through impaired autophagy flux. Diclofenac may be a candidate therapeutic drug in certain type of cancers by inhibiting microtubule-mediated cellular events in combination with clinically utilized nucleoside metabolic inhibitors, including 5-fluorouracil, to block cancer cell proliferation.

Introduction: Neoadjuvant endocrine therapy (NAE) offers a breast-conserving surgery rate and clinical response rate similar to those of neoadjuvant chemotherapy (NAC), while presenting fewer adverse events and lower pathological complete response rates. The assessment of pathological response determines degenerative changes and predicts the prognosis of breast cancer treated with NAC. This study clarified the degenerative changes occurring in breast cancer following NAE., Methods: Our study encompassed two groups: NAE, consisting of 15 patients, and NAC, comprising 18 patients. Tissue samples were obtained from core needle biopsies and surgeries. Nuclear and cell areas were calculated using Autocell analysis. Furthermore, we assessed markers associated with microtubule depolymerization (KIF2A) and initiators of apoptosis (caspase-9)., Results: In the NAC group, we observed significant increases in both cytoplasmic and cell areas. These changes in cytoplasm and cells were notably more pronounced in the NAC group compared to the NAE group. After treatment, KIF2A exhibited a decrease, with the magnitude of change being greater in the NET group than in the NAC group. However, no discernible differences were found in caspase-9 expression between the two groups., Conclusion: Our findings indicate that NAE induces condensation in cancer cells via cell cycle arrest or apoptosis. Conversely, NAC leads to cell enlargement due to the absence of microtubule depolymerization. These discrepancies underscore the importance of accounting for these distinctions when establishing criteria for evaluating pathological responses., (© 2024 The Author(s). Published by S. Karger AG, Basel.)

The structure of the isothiocyanates (ITCs)—erucin, sulforaphane, erysolin, sulforaphene, and phenethyl isothiocyanate—were assessed as well as their respective in vitro anticancer activity on the hepatocellular carcinoma cell line HepG2. All of these ITCs induced both apoptotic and necrotic cell death. FTIR analysis indicated that the ITCs caused changes in cellular components comparable to vinblastine. Despite no observable effect on DNA, the ITCs all induced generation of intracellular reactive oxygen species (ROS) and suppressed microtubule polymerization. The variation in sulfur oxidation states and the presence of an aromatic ring on the ITC side chain affected microtubule depolymerization and intracellular ROS generation, leading to apoptotic and necrotic cancer cell death. Knowing the influences of structural variations of the ITC side chain would be useful for selecting the more potent ITCs ( i.e ., erysolin) for the design and development of effective chemopreventive agents. [ABSTRACT FROM AUTHOR]

Rabies virus (RABV) is the cause of rabies, and is associated with severe neurological symptoms, high mortality rate, and a serious threat to human health. Although cellular tubulin has recently been identified to be incorporated into RABV particles, the effects of RABV infection on the microtubule cytoskeleton remain poorly understood. In this study, we show that RABV infection induces microtubule depolymerization as observed by confocal microscopy, which is closely associated with the formation of the filamentous network of the RABV M protein. Depolymerization of microtubules significantly increases viral RNA synthesis, while the polymerization of microtubules notably inhibits viral RNA synthesis and prevents the viral M protein from inducing the formation of the filamentous network. Furthermore, the histone deacetylase 6 (HDAC6) expression level progressively increases during RABV infection, and the inhibition of HDAC6 deacetylase activity significantly decreases viral RNA synthesis. In addition, the expression of viral M protein alone was found to significantly upregulate HDAC6 expression, leading to a substantial reduction in its substrate, acetylated α-tubulin, eventually resulting in microtubule depolymerization. These results demonstrate that HDAC6 plays a positive role in viral transcription and replication by inducing microtubule depolymerization during RABV infection.

It is widely known that stimulator of interferon genes (STING) can trigger nuclear factor κB (NF-κB) signaling. However, whether and how the NF-κB pathway affects STING signaling remains largely unclear. Here, we report that Toll-like receptor (TLR)-, interleukin-1 receptor (IL-1R)-, tumor necrosis factor receptor (TNFR)-, growth factor receptor (GF-R)-, and protein kinase C (PKC)-mediated NF-κB signaling activation dramatically enhances STING-mediated immune responses. Mechanistically, we find that STING interacts with microtubules, which plays a crucial role in STING intracellular trafficking. We further uncover that activation of the canonical NF-κB pathway induces microtubule depolymerization, which inhibits STING trafficking to lysosomes for degradation. This leads to increased levels of activated STING that persist for a longer period of time. The synergy between NF-κB and STING triggers a cascade-amplified interferon response and robust host antiviral defense. In addition, we observe that several gain-of-function mutations of STING abolish the microtubule-STING interaction and cause abnormal STING trafficking and ligand-independent STING autoactivation. Collectively, our data demonstrate that NF-κB activation enhances STING signaling by regulating microtubule-mediated STING trafficking. [Display omitted] • Activation of a wide range of NF-κB pathways enhances STING signaling responses • NF-κB activation prevents STING degradation and thus enhances STING signaling • NF-κB activation blocks STING turnoff by altering microtubule-based STING transport • Damaged STING-microtubule interaction induces STING hyperactivation Zhang et al. find that activation of a wide range of NF-κB pathways enhances STING-mediated immune responses. NF-κB activation blocks STING degradation by inhibiting microtubule-mediated STING trafficking from the Golgi apparatus to lysosomes and thus prolongs and increases STING signaling. This then augments STING-mediated interferon responses and host antiviral defenses. [ABSTRACT FROM AUTHOR]

A XMAP215/Stu2/Alp14 polymerase can catalyze processively the tubulin addition to the microtubule (MT) plus end. In this work, a model is proposed for the underlying molecular mechanism of the polymerase activity, where the polymerase can not only catalyze processively the tubulin addition to but also promote the tubulin removal from the MT plus end. Based on the model the dynamics of both the wild-type and mutant polymerases is studied theoretically, explaining consistently and well various available experimental data. To further test the model, predicted results are provided., (© 2022 Wiley Periodicals LLC.)

The precise regulation of microtubule dynamics is essential during cell division. The kinesin-13 motor protein MCAK is a potent microtubule depolymerase. The divergent non-motor regions flanking the ATPase domain are critical in regulating its targeting and activity. However, the molecular basis for the function of the non-motor regions within the context of full-length MCAK is unknown. Here, we determine the structure of MCAK motor domain bound to its regulatory C-terminus. Our analysis reveals that the MCAK C-terminus binds to two motor domains in solution and is displaced allosterically upon microtubule binding, which allows its robust accumulation at microtubule ends. These results demonstrate that MCAK undergoes long-range conformational changes involving its C-terminus during the soluble to microtubule-bound transition and that the C-terminus-motor interaction represents a structural intermediate in the MCAK catalytic cycle. Together, our work reveals intrinsic molecular mechanisms underlying the regulation of kinesin-13 activity.

Highlights: [•] Mcp1p is a novel microtubule plus-tip protein tracking both growing and shortening microtubules. [•] Mcp1p is required for microtubule destabilization at cell tips. [•] The kinesin-8 klp6p transports mcp1p to microtubule plus ends. [Copyright &y& Elsevier]

The aim of this study is to investigate the effect of microtubule depolymerization by colchicine on hyperalgesia mediated by transient receptor potential vanilloid 4 (TRPV4) in a neuropathic pain model of chronic compression of the dorsal root ganglion (DRG) (hereafter termed CCD) in rat. Intrathecal administration of microtubule-depolymerizing agent, colchicine, attenuated the activated effect of 4alphaphorbol 12, 13-didecanoate (4alpha-PDD, TRPV4 specific agonist) on mechanical and thermal hyperalgesia in CCD rats. This observation is in agreement with our in vitro experiments with DRG cells that showed a significant attenuation of 4alpha-PDD-activated Ca2+-influx and substance P (SP) release with the colchicine treatment. We conclude that microtubule depolymerization by colchicine can regulate pain sensitivity by depressing the hyperalgesia mediated by TRPV4. [ABSTRACT FROM AUTHOR]

The activity of a kinesin is largely determined by the approximately 350 residue motor domain, and this region alone is sufficient to classify a kinesin as a member of a particular family. The kinesin-13 family are a group of microtubule depolymerizing kinesins and are vital regulators of microtubule length. Kinesin-13s are critical to spindle assembly and chromosome segregation in both mitotic and meiotic cell division and play crucial roles in cilium length control and neuronal development. To better understand the evolution of microtubule depolymerization activity, we created a synthetic ancestral kinesin-13 motor domain. This phylogenetically inferred ancestral motor domain is the sequence predicted to have existed in the common ancestor of the kinesin-13 family. Here we show that the ancestral kinesin-13 motor depolymerizes stabilized microtubules faster than any previously tested depolymerase. This potent activity is more than an order of magnitude faster than the most highly studied kinesin-13, MCAK and allows the ancestral kinesin-13 to depolymerize doubly stabilized microtubules and cause internal breaks within microtubules. These data suggest that the ancestor of the kinesin-13 family was a 'super depolymerizer' and that members of the kinesin-13 family have evolved away from this extreme depolymerizing activity to provide more controlled microtubule depolymerization activity in extant cells.

Microtubule kinetochore attachments are essential for accurate mitosis, but how these force-generating connections move chromosomes remains poorly understood. Processive motion at shortening microtubule ends can be reconstituted in vitro using microbeads conjugated to the budding yeast kinetochore protein Dam1, which forms microtubule- encircling rings. Here, we report that, when Dam1 is linked to a bead cargo by elongated protein tethers, the maximum force transmitted from a disassembling microtubule increases sixfold compared with a short tether. We interpret this significant improvement with a theory that considers the geometry and mechanics of the microtubule-ring-bead system. Our results show the importance of fibrillar links in tethering microtubule ends to cargo: fibrils enable the cargo to align coaxially with the microtubule, thereby increasing the stability of attachment and the mechanical work that it can do. The force-transducing characteristics of fibril-tethered Dam1 are similar to the analogous properties of purified yeast kinetochores, suggesting that a tethered Dam1 ring comprises the main force-bearing unit of the native attachment. [ABSTRACT FROM AUTHOR]

Abstract: A series of novel enediyne-containing molecules, 1-aryl-6-(3,4,5-trimethoxyphenyl)-3(Z)-hexen-1,5-diynes, were synthesized and displayed significant IC50 values of 10−7 to 10−6 M against various cancer cell lines. Of these compounds, 1-(2-pyridinyl)-6-(3,4,5-trimethoxyphenyl)-3(Z)-hexen-1,5-diyne (8) demonstrated the greatest growth inhibition activity. Compound 8 also arrested cancer cells in the G2/M phase and induced apoptosis via activation of Caspase-3. In addition to the G2/M block, compound 8 caused microtubule depolymerization at low concentrations and markedly decreased tumor size in xenographic studies. [Copyright &y& Elsevier]

Abstract: The aim of this study was to investigate the effect of colchicine-induced microtubule depolymerization on allodynia in rats with chronic compression of the dorsal root ganglion (DRG) (CCD) and the effect of colchicine on transient receptor potential vanilloid 4 (TRPV4). Intrathecal administration of the anti-microtubule agent, colchicine, resulted in a dose-dependent and partial reduction in CCD-induced mechanical and thermal allodynia. The reduction of allodynia was associated with significant and dose-dependent decreases in the levels of both TRPV4 mRNA and protein expression in CCD rats. In addition, colchicine resulted in reduction and advance of TRPV4 currents in both DRG neurons and HEK293-TRPV4 cells. The current–voltage (IV) relation in HEK293-TRPV4 cells that were exposed to colchicine displayed a typical outward rectification characteristic of TRPV4 with the reversal potential shifted toward a more positive voltage. In conclusion, intrathecal administration of colchicine attenuated allodynia and TRPV4 contributed to the colchicine-induced attenuation of allodynia in CCD rats. [Copyright &y& Elsevier]

Microtubule targeting agents (MTAs) are widely used cancer chemotherapeutics which conventionally exert their effects during mitosis, leading to mitotic or postmitotic death. However, accumulating evidence suggests that MTAs can also generate death signals during interphase, which may represent a key mechanism in the clinical setting. We reported previously that vincristine and other microtubule destabilizers induce death not only in M phase but also in G1 phase in primary acute lymphoblastic leukemia cells. Here, we sought to investigate and compare the pathways responsible for phase-specific cell death. Primary acute lymphoblastic leukemia cells were subjected to centrifugal elutriation, and cell populations enriched in G1 phase (97%) or G2/M phases (80%) were obtained and treated with vincristine. We found death of M phase cells was associated with established features of mitochondrial-mediated apoptosis, including Bax activation, loss of mitochondrial transmembrane potential, caspase-3 activation, and nucleosomal DNA fragmentation. In contrast, death of G1 phase cells was not associated with pronounced Bax or caspase-3 activation but was associated with loss of mitochondrial transmembrane potential, parylation, nuclear translocation of apoptosis-inducing factor and endonuclease G, and supra-nucleosomal DNA fragmentation, which was enhanced by inhibition of autophagy. The results indicate that microtubule depolymerization induces distinct cell death pathways depending on during which phase of the cell cycle microtubule perturbation occurs. The observation that a specific type of drug can enter a single cell type and induce two different modes of death is novel and intriguing. These findings provide a basis for advancing knowledge of clinical mechanisms of MTAs., Competing Interests: Conflict of interest The authors declare they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

To investigate the different inhibition effects of different sequential usages of microtubule depolymerization drug and polymerization drug on tumor cells. Three tumor cell lines including MCF-7, SK-OV3, A549 were incubated with paclitaxel (PTX) and/or vinorelbine (NVB) of different concentrations. The cyto-toxicity was examined by MTT test after incubating 72 h. According to different drugs and different sequences added to 96-well tissue culture plates, 5 groups were divided: PTX group (Group 1), NVB group (Group 2), PTX plus NVB group (Group 3), PTX first and NVB 4-h-later group (Group 4), and NVB first and PTX 4-h-later group (Group 5). Drug concentrations were 100% peak plasma concentration (PPC), 50% PPC, 25% PPC, 12.5% PPC, and 6. 25% PPC. The inhibition effects on the three tumor cell lines in Group 5 were stronger than those in the other four groups ( P < 0.01). And the inhibition effects in Group 4 were not stronger than those in Groups 1, 2 or 3 ( P > 0.1). Using microtubule depolymerization drug first and then using microtubule polymerization drug has synergic inhibition effect on tumor cells. [ABSTRACT FROM AUTHOR]

Abstract: Gambogic acid (GA), an ingredient isolated from Garcinia hanburyi, has potent anticancer activity both in vitro and in vivo. In the present study, we examined the effects of GA on intracellular microtubules and reconstituted microtubules in vitro. Immunofluorescence microscopy revealed that 2.5 μM GA caused microtubule cytoskeleton disruption and microtubule depolymerization in human breast carcinoma MCF-7 cells, thereby reducing the amount of polymer form of tubulin and increasing the amount of monomer form of tubulin. We further confirmed that GA could depolymerize microtubule associated protein (MAP)-free microtubules and MAP-rich microtubules in vitro. Thus we suggested that GA-induced G2/M phase cell cycle arrest may be attributed to its depolymerization of microtubules. We also revealed that phosphorylation levels of p38 and c-Jun N-terminal kinase-1 (JNK-1) were increased markedly by GA, resulting in apoptosis of MCF-7 cells. Taken together, our results suggested that GA depolymerized microtubules and elevated the phosphorylation levels of JNK1 and p38, which caused G2/M cell cycle arrest and apoptosis in MCF-7 cells. [Copyright &y& Elsevier]

This study tests the hypothesis that microtubule (MT) depolymerization facilitates contraction of rat aorta via activation of Rho-kinase. Aortic rings from Sprague–Dawley rats were placed in a muscle bath for the measurement of isometric force generation. Bath temperature was decreased from 37 to 10–20 °C (30 min), inducing MT depolymerization. Some vessels were treated with nocodazole (10−5 M) or colchicine (10−8–10−5 M) to stabilize the MTs in the depolymerized state, and the remaining vessels were treated with dimethyl sulfoxide (DMSO: vehicle). Warming of vessels to 37 °C induced a significantly greater contraction in nocodazole- and colchicine-treated vessels as compared with controls, and this increase was blocked by pretreatment with taxol (10−5 M; a MT stabilizing agent) [force (mg): NOC 1159±93; COL 1138±69; DMSO 578±14; TAX+NOC 526±43; TAX+COL 538±90]. Following the sustained contraction in response to rewarming, Rho-kinase inhibition with Y-27632 (10−5 M) relaxed nocodazole- and colchicine-treated rings to a significantly greater extent as compared to DMSO-treated vessels (percent relaxation: NOC 64±2; COL 65±5; DMSO 33±5). These results support the hypothesis that MT depolymerization facilitates contraction of rat aorta via activation of Rho-kinase. [Copyright &y& Elsevier]

One of the hallmarks of Alzheimer's disease (AD) is the abnormal state of tau. It is both highly phosphorylated and aggregated into paired helical filaments (PHFs) in neurofibrillary tangles (NFTs). However, the mechanism underlying the hyperphosphorylation of tau in NFTs and neuronal degeneration in AD remains to be elucidated. The fact that hyperphosphorylation of tau in NFTs are also found in the patients with Niemann–Pick disease, type C (NPC), which is a cholesterol storage disease associated with defective intracellular trafficking of exogenous cholesterol, implies that perturbation of cholesterol metabolism may be involved in tau phosphorylation and neurodegeneration. Here, we report that cholesterol deficiency induced by inhibition of cholesterol biosynthesis in cultured neurons results in hyperphosphorylation of tau, accompanied by axonal degeneration associated with microtubule depolymerization. These changes were prevented by concurrent treatment with β-migrating very low-density lipoprotein (β-VLDL) or cholesterol. We propose that intracellular cholesterol plays an essential role in the modulation of tau phosphorylation and the maintenance of microtubule stability. [ABSTRACT FROM AUTHOR]