Stabilization of actin bundles by a dynamin 1/cortactin ring complex is necessary for growth cone filopodia.

Dynamin GTPase, a key molecule in endocytosis, mechanically severs the invaginated membrane upon GTP hydrolysis. Dynamin functions also in regulating actin cytoskeleton, but the mechanisms are yet to be defined. Here we show that dynamin 1, a neuronal isoform of dynamin, and cortactin form ring complexes, which twine around F-actin bundles and stabilize them. By negative-staining EM, dynamin 1-cortactin complexes appeared as "open" or "closed" rings depending on guanine nucleotide conditions. By pyrene actin assembly assay, dynamin 1 stimulated actin assembly in mouse brain cytosol. In vitro incubation of F-actin with both dynamin 1 and cortactin led to the formation of long and thick actin bundles, on which dynamin 1 and cortactin were periodically colocalized in puncta. A depolymerization assay revealed that dynamin 1 and cortactin increased the stability of actin bundles, most prominently in the presence of GTP. In rat cortical neurons and human neuroblastoma cell line, SH-SY5Y, both dynamin 1 and cortactin localized on actin filaments and the bundles at growth cone filopodia as revealed by immunoelectron microscopy. In SH-SY5Y cell, acute inhibition of dynamin 1 by application of dynamin inhibitor led to growth cone collapse. Cortactin knockdown also reduced growth cone filopodia. Together, our results strongly suggest that dynamin 1 and cortactin ring complex mechanically stabilizes F-actin bundles in growth cone filopodia. Thus, the GTPase-dependent mechanochemical enzyme property of dynamin is commonly used both in endocytosis and regulation of F-actin bundles by a dynamin 1-cortactin complex.

Expression of a dynamin 2 mutant associated with Charcot-Marie-Tooth disease leads to aberrant actin dynamics and lamellipodia formation.

Specific mutations in dynamin 2 are linked to Charcot-Marie-Tooth disease (CMT), an inherited peripheral neuropathy. However, the effects of these mutations on dynamin function, particularly in relation to the regulation of the actin cytoskeleton remain unclear. Here, selected CMT-associated dynamin mutants were expressed to examine their role in the pathogenesis of CMT in U2OS cells. Ectopic expression of the dynamin CMT mutants 555Δ3 and K562E caused an approximately 50% decrease in serum stimulation-dependent lamellipodia formation; however, only K562E caused aberrations in the actin cytoskeleton. Immunofluorescence analysis showed that the K562E mutation resulted in the disappearance of radially aligned actin bundles and the simultaneous appearance of F-actin clusters. Live-cell imaging analyses showed F-actin polymers of decreased length assembled into immobile clusters in K562E-expressing cells. The K562E dynamin mutant colocalized with the F-actin clusters, whereas its colocalization with clathrin-coated pit marker proteins was decreased. Essentially the same results were obtained using another cell line, HeLa and NG108-15 cells. The present study is the first to show the association of dynamin CMT mutations with aberrant actin dynamics and lamellipodia, which may contribute to defective endocytosis and myelination in Schwann cells in CMT.

Targeting of tubulin polymerization and induction of mitotic blockage by Methyl 2-(5-fluoro-2-hydroxyphenyl)-1H-benzo[d]imidazole-5-carboxylate (MBIC) in human cervical cancer HeLa cell.

BACKGROUND: Microtubule Targeting Agents (MTAs) including paclitaxel, colchicine and vinca alkaloids are widely used in the treatment of various cancers. As with most chemotherapeutic agents, adverse effects and drug resistance are commonly associated with the clinical use of these agents. Methyl 2-(5-fluoro-2-hydroxyphenyl)-1H- benzo[d]imidazole-5-carboxylate (MBIC), a benzimidazole derivative displays greater toxicity against various cancer compared to normal human cell lines. The present study, focused on the cytotoxic effects of MBIC against HeLa cervical cancer cells and possible actions on the microtubule assembly. METHODS: Apoptosis detection and cell-cycle assays were performed to determine the type of cell death and the phase of cell cycle arrest in HeLa cells. Tubulin polymerization assay and live-cell imaging were performed to visualize effects on the microtubule assembly in the presence of MBIC. Mitotic kinases and mitochondrial-dependent apoptotic proteins were evaluated by Western blot analysis. In addition, the synergistic effect of MBIC with low doses of selected chemotherapeutic actions were examined against the cancer cells. RESULTS: Results from the present study showed that following treatment with MBIC, the HeLa cells went into mitotic arrest comprising of multi-nucleation and unsegregated chromosomes with a prolonged G2-M phase. In addition, the HeLa cells showed signs of mitochondrial-dependant apoptotic features such as the release of cytochrome c and activation of caspases. MBIC markedly interferes with tubulin polymerization. Western blotting results indicated that MBIC affects mitotic regulatory machinery by up-regulating BubR1, Cyclin B1, CDK1 and down-regulation of Aurora B. In addition, MBIC displayed synergistic effect when given in combination with colchicine, nocodazole, paclitaxel and doxorubicin. CONCLUSION: Taken together, our study demonstrated the distinctive microtubule destabilizing effects of MBIC against cervical cancer cells in vitro. Besides that, MBIC exhibited synergistic effects with low doses of selected anticancer drugs and thus, may potentially reduce the toxicity and drug resistance to these agents.

CLIP-170 tethers kinetochores to microtubule plus ends against poleward force by dynein for stable kinetochore-microtubule attachment.

The cytoplasmic linker protein (CLIP)-170 localizes to kinetochores and is suggested to function in stable attachment of kinetochores to microtubule ends. Here we show that defects in kinetochore-microtubule attachment and chromosome alignment in CLIP-170-depleted cells were rescued by co-depletion of p150glued, a dynactin subunit required for kinetochore localization of CLIP-170. CLIP-170 recruited p150glued to microtubule ends. Kinetochore localization at microtubule ends was perturbed by CLIP-170 depletion, which was rescued by co-depleting p150glued. Our results imply that CLIP-170 tethers kinetochores to microtubule ends against the dynein-mediated poleward force to slide kinetochores along microtubules, facilitating the stable kinetochore attachment to microtubules.