Role of microtubule dynamics in Wallerian degeneration and nerve regeneration after peripheral nerve injury

Wallerian degeneration,the progressive disintegration of distal axons and myelin that occurs after peripheral nerve injury,is essential for creating a permissive microenvironment for nerve regeneration,and involves cytoskeletal reconstruction.However,it is unclear whether microtubule dynamics play a role in this process.To address this,we treated cultured sciatic nerve explants,an in vitro model of Wallerian degeneration,with the microtubule-targeting agents paclitaxel and nocodazole.We found that paclitaxel-induced microtubule stabilization promoted axon and myelin degeneration and Schwann cell dedifferentiation,whereas nocodazole-induced microtubule destabilization inhibited these processes.Evaluation of an in vivo model of peripheral nerve injury showed that treatment with paclitaxel or nocodazole accelerated or attenuated axonal regeneration,as well as functional recovery of nerve conduction and target muscle and motor behavior,respectively.These results suggest that microtubule dynamics participate in peripheral nerve regeneration after injury by affecting Wallerian degeneration.This study was approved by the Animal Care and Use Committee of Southern Medical University,China(approval No.SMUL2015081) on October 15,2015.

Stathmin destabilizing microtubule dynamics promotes malignant potential in cancer cells by epithelial-mesenchymal transition

BACKGROUND： Stathmin is a ubiquitous cytosolic regulatory phosphoprotein and is overexpressed in different human malignancies. The main physiological function of stathmin is to interfere with microtubule dynamics by promoting depolymerization of microtubules or by preventing polymerization of tubulin heterodimers. Stathmin plays important roles in regulating many cellular functions as a result of its microtubuledestabilizing activity. Currently, the critical roles of stathmin in cancer cells, as well as in lymphocytes have been valued. This review discusses stathmin and microtubule dynamics in cancer development, and hypothesizes their possible relationship with epithelial-mesenchymal transition（EMT）.DATA SOURCES： A PubMed search using such terms as ＂stathmin＂, ＂microtubule dynamics＂, ＂epithelial-mesenchymal transition＂, ＂EMT＂, ＂malignant potential＂ and ＂cancer＂ was performed to identify relevant studies published in English.More than 100 related articles were reviewed.RESULTS： The literature clearly documented the relationship between stathmin and its microtubule-destabilizing activity of cancer development. However, the particular mechanism is poorly understood. Microtubule disruption is essential for EMT, which is a crucial process during cancer development. As a microtubule-destabilizing protein, stathmin may promote malignant potential in cancer cells by initiating EMT.CONCLUSIONS： We propose that there is a stathminmicrotubule dynamics-EMT（S-M-E） axis during cancer development. By this axis, stathmin together with itsmicrotubule-destabilizing activity contributes to EMT, which stimulates the malignant potential in cancer cells.

Microtubule dynamics in axon guidance

Precise modulation of the cytoskeleton is involved in a variety of cellular processes including cell division, migration, polarity, and adhesion. In developing post-mitotic neurons, extracellular guidance cues not only trigger signaling cascades that act at a distance to indirectly regulate microtubule distribution, and assembly and disassembly in the growth cone, but also directly modulate microtubule stability and dynamics through coupling of guidance receptors with microtubules to control growth-cone turning. Microtubule-associated proteins including classical microtubule-associated proteins and microtubule plus-end tracking proteins are required for modulating microtubule dynamics to influence growth-cone steering. Multiple key signaling components, such as calcium, small GTPases, glycogen synthase kinase-313, and c-Jun N-terminal kinase, link upstream signal cascades to microtubule stability and dynamics in the growth cone to control axon outgrowth and projection. Understanding the functions and regulation of microtubule dynamics in the growth cone provides new insights into the molecular mechanisms of axon guidance.

SUMOylation of α-tubulin is a novel modification regulating microtubule dynamics

Microtubules (MTs) are regulated by a number of known posttranslational modifications (PTMs) on α/β-tubulin to fulfill diverse cellular functions. Here, we showed that SUMOylation is a novel PTM on α-tubulin in vivo and in vitro. The SUMOylation on α-tubulin mainly occurred at Lys 96 (K96), K166, and K304 of soluble α-tubulin and could be removed by small ubiquitin-related modifier (SUMO)-specific peptidase 1. In vitro experiments showed that tubulin SUMOylation could reduce interprotofilament interaction, promote MT catastrophe, and impede MT polymerization. In cells, mutation of the SUMOylation sites on α-tubulin reduced catastrophe frequency and increased the proportion of polymerized α-tubulin, while upregulation of SUMOylation with fusion of SUMO1 reduced α-tubulin assembly into MTs. Additionally, overexpression of SUMOylation-deficient α-tubulin attenuated the neurite extension in Neuro-2a cells. Thus, SUMOylation on α-tubulin represents a new player in the regulation of MT properties.

Fidgetin interacting with microtubule end binding protein EB3 affects axonal regrowth in spinal cord injury

Fidgetin,a microtubule-severing enzyme,regulates neurite outgrowth,axonal regeneration,and cell migration by trimming off the labile domain of microtubule polymers.Because maintenance of the microtubule labile domain is essential for axon initiation,elongation,and navigation,it is of interest to determine whether augmenting the microtubule labile domain via depletion of fidgetin serves as a therapeutic approach to promote axonal regrowth in spinal cord injury.In this study,we constructed rat models of spinal cord injury and sciatic nerve injury.Compared with spinal cord injury,we found that expression level of tyrosinated microtubules in the labile portion of microtubules continuously increased,whereas fidgetin decreased after peripheral nerve injury.Depletion of fidgetin enhanced axon regeneration after spinal cord injury,whereas expression level of end binding protein 3(EB3)markedly increased.Next,we performed RNA interference to knockdown EB3 or fidgetin.We found that deletion of EB3 did not change fidgetin expression.Conversely,deletion of fidgetin markedly increased expression of tyrosinated microtubules and EB3.Deletion of fidgetin increased the amount of EB3 at the end of neurites and thereby increased the level of tyrosinated microtubules.Finally,we deleted EB3 and overexpressed fidgetin.We found that fidgetin trimmed tyrosinated tubulins by interacting with EB3.When fidgetin was deleted,the labile portion of microtubules was elongated,and as a result the length of axons and number of axon branches were increased.These findings suggest that fidgetin can be used as a novel therapeutic target to promote axonal regeneration after spinal cord injury.Furthermore,they reveal an innovative mechanism by which fidgetin preferentially severs labile microtubules.

ARK2 stabilizes the plus-end of microtubules and promotes microtubule bundling in Arabidopsis

Microtubule dynamics and organization are important for plant cell morphogenesis and development.The microtubule-based motor protein kinesins are mainly responsible for the transport of some organelles and vesicles,although several have also been shown to regulate microtubule organization.The ARMADILLO REPEAT KINESIN(ARK)family is a plant-specific motor protein subfamily that consists of three members(ARK1,ARK2,and ARK3)in Arabidopsis thaliana.ARK2 has been shown to participate in root epidermal cell morphogenesis.However,whether and how ARK2 associates with microtubules needs further elucidation.Here,we demonstrated that ARK2 co-localizes with microtubules and facilitates microtubule bundling in vitro and in vivo.Pharmacological assays and microtubule dynamics analyses indicated that ARK2 stabilizes cortical microtubules.Live-cell imaging revealed that ARK2moves along cortical microtubules in a processive mode and localizes both at the plus-end and the sidewall of microtubules.ARK2 therefore tracks and stabilizes the growing plus-ends of microtubules,which facilitates the formation of parallel microtubule bundles.

α1A and α1C form microtubules to display distinct properties mainly mediated by their C-terminal tails

Microtubules consisting of a/b-tubulin dimers play critical roles in cells.More than seven genes encode a-tubulin in vertebrates.However,the property of microtubules composed of different a-tubulin isotypes is largely unknown.Here,we purified recombinant tubulin heterodimers of mouse a-tubulin isotypes including a1A and a1C with b-tubulin isotype b2A.In vitro microtubule reconstitution assay detected that a1C/b2A microtubules grew faster and underwent catastrophe less frequently than a1A/b2A microtubules.Generation of chimeric tail-swapped and point-mutation tubulins revealed that the carboxyl-terminal(C-terminal)tails of a-tubulin isotypes largely accounted for the differences in polymerization dynamics of a1A/b2A and a1C/b2A microtubules.Kinetics analysis showed that in comparison to a1A/b2A microtubules,a1C/b2A microtubules displayed higher on-rate,lower off-rate,and similar GTP hydrolysis rate at the plus-end,suggesting a contribution of higher plus-end affinity to faster growth and less frequent catastrophe of a1C/b2A microtubules.Furthermore,EB1 had a higher binding ability to a1C/b2A microtubules than to a1A/b2A ones,which could also be attributed to the difference in the C-terminal tails of these two a-tubulin isotypes.Thus,a-tubulin isotypes diversify microtubule properties,which,to a great extent,could be accounted by their C-terminal tails.

High-speed image reconstruction for optically sectioned,super-resolution structured illumination microscopy

Super-resolution structured illumination microscopy(SR-SIM)is an outstanding method for visualizing the subcellular dynamics in living cells.To date,by using elaborately designed systems and algorithms,SR-SIM can achieve rapid,optically sectioned,SR observation with hundreds to thousands of time points.However,real-time observation is still out of reach for most SIM setups as conventional algorithms for image reconstruction involve a heavy computing burden.To address this limitation,an accelerated reconstruction algorithm was developed by implementing a simplified workflow for SR-SIM,termed joint space and frequency reconstruction.This algorithm results in an 80-fold improvement in reconstruction speed relative to the widely used Wiener-SIM.Critically,the increased processing speed does not come at the expense of spatial resolution or sectioning capability,as demonstrated by live imaging of microtubule dynamics and mitochondrial tubulation.

Redox-dependent regulation of end-binding protein 1 activity by glutathionylation

Cytoskeletal proteins are susceptible to glutathionylation under oxidizing conditions,and oxidative damage has been implicated in several neurodegenerative diseases.End-binding protein 1(EB1)is a master regulator of microtubule plus-end tracking proteins(+TIPs)and is critically involved in the control of microtubule dynamics and cellular processes.However,the impact of glutathionylation on EB1 functions remains unknown.Here we reveal that glutathionylation is important for controlling EB1 activity and protecting EB1 from irreversible oxidation.In vitro biochemical and cellular assays reveal that EB1 is glutathionylated.Diamide,a mild oxidizing reagent,reduces EB1 comet number and length in cells,indicating the impairment of microtubule dynamics.Three cysteine residues of EB1 are glutathionylated,with mutations of these three cysteines to serines attenuating microtubule dynamics but buffering diamide-induced decrease in microtubule dynamics.In addition,glutaredoxin 1(Grx1)deglutathionylates EB1,and Grx1 depletion suppresses microtubule dynamics and leads to defects in cell division orientation and cell migration,suggesting a critical role of Grx1-mediated deglutathionylation in maintaining EB1 activity.Collectively,these data reveal that EB1 glutathionylation is an important protective mechanism for the regulation of microtubule dynamics and microtubule-based cellular activities.