SUMOylation of microtubule-cleaving enzyme KATNA1 promotes microtubule severing and neurite outgrowth.

Katanin p60 ATPase-containing subunit A1 (KATNA1) is a microtubule-cleaving enzyme that regulates the development of neural protrusions through cytoskeletal rearrangements. However, the mechanism underlying the linkage of the small ubiquitin-like modifier (SUMO) protein to KATNA1 and how this modification regulates the development of neural protrusions is unclear. Here we discovered, using mass spectrometry analysis, that SUMO-conjugating enzyme UBC9, an enzyme necessary for the SUMOylation process, was present in the KATNA1 interactome. Moreover, GST-pull down and co-immunoprecipitation assays confirmed that KATNA1 and SUMO interact. We further demonstrated using immunofluorescence experiments that KATNA1 and the SUMO2 isoform colocalized in hippocampal neurites. We also performed a bioinformatics analysis of KATNA1 protein sequences to identify three potentially conserved SUMOylation sites (K77, K157, and K330) among vertebrates. Mutation of K330, but not K77 or K157, abolished KATNA1-induced microtubule severing and decreased the level of binding observed for KATNA1 and SUMO2. Cotransfection of SUMO2 and wildtype KATNA1 in COS7 cells increased microtubule severing, whereas no effect was observed after cotransfection with the K330R KATNA1 mutant. Furthermore, in cultured hippocampal neurons, overexpression of wildtype KATNA1 significantly promoted neurite outgrowth, whereas the K330R mutant eliminated this effect. Taken together, our results demonstrate that the K330 site in KATNA1 is modified by SUMOylation and SUMOylation of KATNA1 promotes microtubule dynamics and hippocampal neurite outgrowth.

Microtubular TRIM36 E3 Ubiquitin Ligase in Embryonic Development and Spermatogenesis.

TRIM36 is a member of the tripartite motif (TRIM) family of RING-containing proteins, also known as Haprin, which was first discovered for its abundance in testis and found to be implicated in the spermatozoa acrosome reaction. TRIM36 is a microtubule-associated E3 ubiquitin ligase that plays a role in cytoskeletal organization, and according to data gathered in different species, coordinates growth speed and stability, acting on the microtubules' plus end, and impacting on cell cycle progression. TRIM36 is also crucial for early developmental processes, in Xenopus, where it is needed for dorso-ventral axis formation, but also in humans as bi-allelic mutations in the TRIM36 gene cause a form of severe neural tube closure defect, called anencephaly. Here, we review TRIM36-related mechanisms implicated in such composite physiological and pathological processes.

WDR62 localizes katanin at spindle poles to ensure synchronous chromosome segregation.

Mutations in the WDR62 gene cause primary microcephaly, a pathological condition often associated with defective cell division that results in severe brain developmental defects. The precise function and localization of WDR62 within the mitotic spindle is, however, still under debate, as it has been proposed to act either at centrosomes or on the mitotic spindle. Here we explored the cellular functions of WDR62 in human epithelial cell lines using both short-term siRNA protein depletions and long-term CRISPR/Cas9 gene knockouts. We demonstrate that WDR62 localizes at spindle poles, promoting the recruitment of the microtubule-severing enzyme katanin. Depletion or loss of WDR62 stabilizes spindle microtubules due to insufficient microtubule minus-end depolymerization but does not affect plus-end microtubule dynamics. During chromosome segregation, WDR62 and katanin promote efficient poleward microtubule flux and favor the synchronicity of poleward movements in anaphase to prevent lagging chromosomes. We speculate that these lagging chromosomes might be linked to developmental defects in primary microcephaly.

Small GTPases modulate intrinsic and extrinsic forces that control epithelial folding in Drosophila embryos.

Epithelial folding is a common means to execute morphogenetic movements. The gastrulating Drosophila embryo offers many examples of epithelial folding events, including the ventral, cephalic, and dorsal furrows. Each of these folding events is associated with changes in intracellular contractility and/or cytoskeleton structures that autonomously promote epithelial folding. Here, we review accumulating evidence that suggests the progression and final form of ventral, cephalic, and dorsal furrows are also influenced by the behaviour of cells neighbouring these folds. We further discuss the prevalence and importance of junctional rearrangements during epithelial folding events, suggesting adherens junction components are prime candidates to modulate the transmission of the intercellular forces that influence folding events. Finally, we discuss how recently developed methods that enable precise spatial and/or temporal control of protein activity allow direct testing of molecular models of morphogenesis in vivo.

NEAT1 regulates microtubule stabilization via FZD3/GSK3ß/P-tau pathway in SH-SY5Y cells and APP/PS1 mice.

Nuclear paraspeckles assembly transcript 1 (NEAT1) is a well-known long noncoding RNA (lncRNA) with various functions in different physiological and pathological processes. Notably, aberrant NEAT1 expression is implicated in the pathogenesis of various neurodegenerative diseases, including Alzheimer's disease (AD). However, the molecular mechanism of NEAT1 in AD remains poorly understood. In this study, we investigated that NEAT1 regulated microtubules (MTs) polymerization via FZD3/GSK3ß/p-tau pathway. Downregulation of NEAT1 inhibited Frizzled Class Receptor 3 (FZD3) transcription activity by suppressing H3K27 acetylation (H3K27Ac) at the FZD3 promoter. Our data also demonstrated that P300, an important histone acetyltransferases (HAT), recruited by NEAT1 to bind to FZD3 promoter and mediated its transcription via regulating histone acetylation. In addition, according to immunofluorescence staining of MTs, metformin, a medicine for the treatment of diabetes mellitus, rescued the reduced length of neurites detected in NEAT1 silencing cells. We suspected that metformin may play a neuroprotective role in early AD by increasing NEAT1 expression and through FZD3/GSK3ß/p-tau pathway. Collectively, NEAT1 regulates microtubule stabilization via FZD3/GSK3ß/P-tau pathway and influences FZD3 transcription activity in the epigenetic way.

Polar relaxation by dynein-mediated removal of cortical myosin II.

Nearly six decades ago, Lewis Wolpert proposed the relaxation of the polar cell cortex by the radial arrays of astral microtubules as a mechanism for cleavage furrow induction. While this mechanism has remained controversial, recent work has provided evidence for polar relaxation by astral microtubules, although its molecular mechanisms remain elusive. Here, using C. elegans embryos, we show that polar relaxation is achieved through dynein-mediated removal of myosin II from the polar cortexes. Mutants that position centrosomes closer to the polar cortex accelerated furrow induction, whereas suppression of dynein activity delayed furrowing. We show that dynein-mediated removal of myosin II from the polar cortexes triggers a bidirectional cortical flow toward the cell equator, which induces the assembly of the actomyosin contractile ring. These results provide a molecular mechanism for the aster-dependent polar relaxation, which works in parallel with equatorial stimulation to promote robust cytokinesis.

PLK1 plays dual roles in centralspindlin regulation during cytokinesis.

Cytokinesis begins upon anaphase onset. An early step involves local activation of the small GTPase RhoA, which triggers assembly of an actomyosin-based contractile ring at the equatorial cortex. Here, we delineated the contributions of PLK1 and Aurora B to RhoA activation and cytokinesis initiation in human cells. Knock-down of PRC1, which disrupts the spindle midzone, revealed the existence of two pathways that can initiate cleavage furrow ingression. One pathway depends on a well-organized spindle midzone and PLK1, while the other depends on Aurora B activity and centralspindlin at the equatorial cortex and can operate independently of PLK1. We further show that PLK1 inhibition sequesters centralspindlin onto the spindle midzone, making it unavailable for Aurora B at the equatorial cortex. We propose that PLK1 activity promotes the release of centralspindlin from the spindle midzone through inhibition of PRC1, allowing centralspindlin to function as a regulator of spindle midzone formation and as an activator of RhoA at the equatorial cortex.

Photo-control of the mitotic kinesin Eg5 using a novel photochromic inhibitor composed of a spiropyran derivative.

In this study, we synthesized a novel photochromic inhibitor of the mitotic kinesin Eg5, which is composed of the photochromic compound spiropyran to photo-control the function of Eg5. The compound (S)-2, 3-dispiropyran propionic acid (DSPPA) exhibits reversible spiropyran-merocyanine photo-isomerization upon irradiation with visible or ultra-violet light. DSPPA induced reversible changes in the inhibitory effect on Eg5 ATPase and motor activities, which correlates with the spiropyran-merocyanine photo-isomerization. Microtubule-dependent ATPase activity was significantly more inhibited by the spiropyran isomer of DSPPA than by the merocyanine isomer. Additionally, an in vitro motility assay revealed that the microtubule gliding velocity was reduced more by the spiropyran isomer than by the merocyanine isomer. This indicates that the spiropyran derivative may be useful in regulating the function of the mitotic kinesin.

Altered chemomechanical coupling causes impaired motility of the kinesin-4 motors KIF27 and KIF7.

Kinesin-4 motors play important roles in cell division, microtubule organization, and signaling. Understanding how motors perform their functions requires an understanding of their mechanochemical and motility properties. We demonstrate that KIF27 can influence microtubule dynamics, suggesting a conserved function in microtubule organization across the kinesin-4 family. However, kinesin-4 motors display dramatically different motility characteristics: KIF4 and KIF21 motors are fast and processive, KIF7 and its Drosophila melanogaster homologue Costal2 (Cos2) are immotile, and KIF27 is slow and processive. Neither KIF7 nor KIF27 can cooperate for fast processive transport when working in teams. The mechanistic basis of immotile KIF7 behavior arises from an inability to release adenosine diphosphate in response to microtubule binding, whereas slow processive KIF27 behavior arises from a slow adenosine triphosphatase rate and a high affinity for both adenosine triphosphate and microtubules. We suggest that evolutionarily selected sequence differences enable immotile KIF7 and Cos2 motors to function not as transporters but as microtubule-based tethers of signaling complexes.

Plk4 and Aurora A cooperate in the initiation of acentriolar spindle assembly in mammalian oocytes.

Establishing the bipolar spindle in mammalian oocytes after their prolonged arrest is crucial for meiotic fidelity and subsequent development. In contrast to somatic cells, the first meiotic spindle assembles in the absence of centriole-containing centrosomes. Ran-GTP can promote microtubule nucleation near chromatin, but additional unidentified factors are postulated for the activity of multiple acentriolar microtubule organizing centers in the oocyte. We now demonstrate that partially overlapping, nonredundant functions of Aurora A and Plk4 kinases contribute to initiate acentriolar meiosis I spindle formation. Loss of microtubule nucleation after simultaneous chemical inhibition of both kinases can be significantly rescued by drug-resistant Aurora A alone. Drug-resistant Plk4 can enhance Aurora A-mediated rescue, and, accordingly, Plk4 can phosphorylate and potentiate the activity of Aurora A in vitro. Both kinases function distinctly from Ran, which amplifies microtubule growth. We conclude that Aurora A and Plk4 are rate-limiting factors contributing to microtubule growth as the acentriolar oocyte resumes meiosis.

The yeast kinesin-5 Cin8 interacts with the microtubule in a noncanonical manner.

Kinesin motors play central roles in establishing and maintaining the mitotic spindle during cell division. Unlike most other kinesins, Cin8, a kinesin-5 motor in Saccharomyces cerevisiae, can move bidirectionally along microtubules, switching directionality according to biochemical conditions, a behavior that remains largely unexplained. To this end, we used biochemical rate and equilibrium constant measurements as well as cryo-electron microscopy methodologies to investigate the microtubule interactions of the Cin8 motor domain. These experiments unexpectedly revealed that, whereas Cin8 ATPase kinetics fell within measured ranges for kinesins (especially kinesin-5 proteins), approximately four motors can bind each αß-tubulin dimer within the microtubule lattice. This result contrasted with those observations on other known kinesins, which can bind only a single "canonical" site per tubulin dimer. Competition assays with human kinesin-5 (Eg5) only partially abrogated this behavior, indicating that Cin8 binds microtubules not only at the canonical site, but also one or more separate ("noncanonical") sites. Moreover, we found that deleting the large, class-specific insert in the microtubule-binding loop 8 reverts Cin8 to one motor per αß-tubulin in the microtubule. The novel microtubule-binding mode of Cin8 identified here provides a potential explanation for Cin8 clustering along microtubules and potentially may contribute to the mechanism for direction reversal.

Differential effects of the dynein-regulatory factor Lissencephaly-1 on processive dynein-dynactin motility.

Cytoplasmic dynein is the primary minus-end-directed microtubule motor protein in animal cells, performing a wide range of motile activities, including transport of vesicular cargos, mRNAs, viruses, and proteins. Lissencephaly-1 (LIS1) is a highly conserved dynein-regulatory factor that binds directly to the dynein motor domain, uncoupling the enzymatic and mechanical cycles of the motor and stalling dynein on the microtubule track. Dynactin, another ubiquitous dynein-regulatory factor, releases dynein from an autoinhibited state, leading to a dramatic increase in fast, processive dynein motility. How these opposing activities are integrated to control dynein motility is unknown. Here, we used fluorescence single-molecule microscopy to study the interaction of LIS1 with the processive dynein-dynactin-BicD2N (DDB) complex. Surprisingly, in contrast to the prevailing model for LIS1 function established in the context of dynein alone, we found that binding of LIS1 to DDB does not strongly disrupt processive motility. Motile DDB complexes bound up to two LIS1 dimers, and mutational analysis suggested that LIS1 binds directly to the dynein motor domains during DDB movement. Interestingly, LIS1 enhanced DDB velocity in a concentration-dependent manner, in contrast to observations of the effect of LIS1 on the motility of isolated dynein. Thus, LIS1 exerts concentration-dependent effects on dynein motility and can synergize with dynactin to enhance processive dynein movement. Our results suggest that the effect of LIS1 on dynein motility depends on both LIS1 concentration and the presence of other regulatory factors such as dynactin and may provide new insights into the mechanism of LIS1 haploinsufficiency in the neurodevelopmental disorder lissencephaly.

Phosphorylation at serine 31 targets tyrosine hydroxylase to vesicles for transport along microtubules.

Tyrosine hydroxylase (TH) catalyzes the conversion of l-tyrosine into l-DOPA, which is the rate-limiting step in the synthesis of catecholamines, such as dopamine, in dopaminergergic neurons. Low dopamine levels and death of the dopaminergic neurons are hallmarks of Parkinson's disease (PD), where α-synuclein is also a key player. TH is highly regulated, notably by phosphorylation of several Ser/Thr residues in the N-terminal tail. However, the functional role of TH phosphorylation at the Ser-31 site (THSer(P)-31) remains unclear. Here, we report that THSer(P)-31 co-distributes with the Golgi complex and synaptic-like vesicles in rat and human dopaminergic cells. We also found that the TH microsomal fraction content decreases after inhibition of cyclin-dependent kinase 5 (Cdk5) and ERK1/2. The cellular distribution of an overexpressed phospho-null mutant, TH1-S31A, was restricted to the soma of neuroblastoma cells, with decreased association with the microsomal fraction, whereas a phospho-mimic mutant, TH1-S31E, was distributed throughout the soma and neurites. TH1-S31E associated with vesicular monoamine transporter 2 (VMAT2) and α-synuclein in neuroblastoma cells, and endogenous THSer(P)-31 was detected in VMAT2- and α-synuclein-immunoprecipitated mouse brain samples. Microtubule disruption or co-transfection with α-synuclein A53T, a PD-associated mutation, caused TH1-S31E accumulation in the cell soma. Our results indicate that Ser-31 phosphorylation may regulate TH subcellular localization by enabling its transport along microtubules, notably toward the projection terminals. These findings disclose a new mechanism of TH regulation by phosphorylation and reveal its interaction with key players in PD, opening up new research avenues for better understanding dopamine synthesis in physiological and pathological states.

Microtubule minus-end regulation at spindle poles by an ASPM-katanin complex.

ASPM (known as Asp in fly and ASPM-1 in worm) is a microcephaly-associated protein family that regulates spindle architecture, but the underlying mechanism is poorly understood. Here, we show that ASPM forms a complex with another protein linked to microcephaly, the microtubule-severing ATPase katanin. ASPM and katanin localize to spindle poles in a mutually dependent manner and regulate spindle flux. X-ray crystallography revealed that the heterodimer formed by the N- and C-terminal domains of the katanin subunits p60 and p80, respectively, binds conserved motifs in ASPM. Reconstitution experiments demonstrated that ASPM autonomously tracks growing microtubule minus ends and inhibits their growth, while katanin decorates and bends both ends of dynamic microtubules and potentiates the minus-end blocking activity of ASPM. ASPM also binds along microtubules, recruits katanin and promotes katanin-mediated severing of dynamic microtubules. We propose that the ASPM-katanin complex controls microtubule disassembly at spindle poles and that misregulation of this process can lead to microcephaly.

Relay Drug Delivery for Amplifying Targeting Signal and Enhancing Anticancer Efficacy.

A "relay drug delivery" system based on two distinct modules, which is composed of a signal transmission nanocarrier A (NCA ) that can specifically induce tumor blood vessel inflammation generation and an execution biomimetic nanocarrier B (NCB ) that can accumulate at the tumor site by receiving the broadcasting signals generated by NCA , is developed for amplifying active tumor targeting signal and enhancing antitumor therapy.

Ndel1 suppresses ciliogenesis in proliferating cells by regulating the trichoplein-Aurora A pathway.

Primary cilia protrude from the surface of quiescent cells and disassemble at cell cycle reentry. We previously showed that ciliary reassembly is suppressed by trichoplein-mediated Aurora A activation pathway in growing cells. Here, we report that Ndel1, a well-known modulator of dynein activity, localizes at the subdistal appendage of the mother centriole, which nucleates a primary cilium. In the presence of serum, Ndel1 depletion reduces trichoplein at the mother centriole and induces unscheduled primary cilia formation, which is reverted by forced trichoplein expression or coknockdown of KCTD17 (an E3 ligase component protein for trichoplein). Serum starvation induced transient Ndel1 degradation, subsequent to the disappearance of trichoplein at the mother centriole. Forced expression of Ndel1 suppressed trichoplein degradation and axonemal microtubule extension during ciliogenesis, similar to trichoplein induction or KCTD17 knockdown. Most importantly, the proportion of ciliated and quiescent cells was increased in the kidney tubular epithelia of newborn Ndel1-hypomorphic mice. Thus, Ndel1 acts as a novel upstream regulator of the trichoplein-Aurora A pathway to inhibit primary cilia assembly.

ASPP1/2-PP1 complexes are required for chromosome segregation and kinetochore-microtubule attachments.

Regulated interactions between kinetochores and spindle microtubules are critical for maintaining genomic stability during chromosome segregation. Defects in chromosome segregation are widespread phenomenon in human cancers that are thought to serve as the fuel for tumorigenic progression. Tumor suppressor proteins ASPP1 and ASPP2, two members of the apoptosis stimulating proteins of p53 (ASPP) family, are frequently down-regulated in human cancers. Here we report that ASPP1/2 are required for proper mitotic progression. In ASPP1/2 co-depleted cells, the persistence of unaligned chromosomes and the reduction of tension across sister kinetochores on aligned chromosomes resulted in persistent spindle assembly checkpoint (SAC) activation. Using protein affinity purification methods, we searched for functional partners of ASPP1/2, and found that ASPP1/2 were associated with a subset of kinetochore proteins (Hec1, KNL-1, and CENP-F). It was found that ASPP1/2 act as PP1-targeting subunits to facilitate the interaction between PP1 and Hec1, and catalyze Hec1 (Ser165) dephosphorylation during late mitosis. These observations revealed a previously unrecognized function of ASPP1/2 in chromosome segregation and kinetochore-microtubule attachments that likely contributes to their roles in chromosome stability and tumor suppression.

Senescent stromal cells induce cancer cell migration via inhibition of RhoA/ROCK/myosin-based cell contractility.

Cells induced into senescence exhibit a marked increase in the secretion of pro-inflammatory cytokines termed senescence-associated secretory phenotype (SASP). Here we report that SASP from senescent stromal fibroblasts promote spontaneous morphological changes accompanied by an aggressive migratory behavior in originally non-motile human breast cancer cells. This phenotypic switch is coordinated, in space and time, by a dramatic reorganization of the actin and microtubule filament networks, a discrete polarization of EB1 comets, and an unconventional front-to-back inversion of nucleus-MTOC polarity. SASP-induced morphological/migratory changes are critically dependent on microtubule integrity and dynamics, and are coordinated by the inhibition of RhoA and cell contractility. RhoA/ROCK inhibition reduces focal adhesions and traction forces, while promoting a novel gliding mode of migration.

DYNLT (Tctex-1) forms a tripartite complex with dynein intermediate chain and RagA, hence linking this small GTPase to the dynein motor.

It has been suggested that DYNLT, a dynein light chain known to bind to various cellular and viral proteins, can function as a microtubule-cargo adaptor. Recent data showed that DYNLT links the small GTPase Rab3D to microtubules and, for this to occur, the DYNLT homodimer needs to display a binding site for dynein intermediate chain together with a binding site for the small GTPase. We have analysed in detail how RagA, another small GTPase, associates to DYNLT. After narrowing down the binding site of RagA to DYNLT we could identify that a ß strand, part of the RagA G3 box involved in nucleotide binding, mediates this association. Interestingly, we show that both microtubule-associated DYNLT and cytoplasmic DYNLT are equally able to bind to the small GTPases Rab3D and RagA. Using NMR spectroscopy, we analysed the binding of dynein intermediate chain and RagA to mammalian DYNLT. Our experiments identify residues of DYNLT affected by dynein intermediate chain binding and residues affected by RagA binding, hence distinguishing the docking site for each of them. In summary, our results shed light on the mechanisms adopted by DYNLT when binding to protein cargoes that become transported alongside microtubules bound to the dynein motor.

PLK1-dependent activation of LRRK1 regulates spindle orientation by phosphorylating CDK5RAP2.

Correct formation of the cell division axis requires the initial precise orientation of the mitotic spindle. Proper spindle orientation depends on centrosome maturation, and Polo-like kinase 1 (PLK1) is known to play a crucial role in this process. However, the molecular mechanisms that function downstream of PLK1 are not well understood. Here we show that LRRK1 is a PLK1 substrate that is phosphorylated on Ser 1790. PLK1 phosphorylation is required for CDK1-mediated activation of LRRK1 at the centrosomes, and this in turn regulates mitotic spindle orientation by nucleating the growth of astral microtubules from the centrosomes. Interestingly, LRRK1 in turn phosphorylates CDK5RAP2(Cep215), a human homologue of Drosophila Centrosomin (Cnn), in its γ-tubulin-binding motif, thus promoting the interaction of CDK5RAP2 with γ-tubulin. LRRK1 phosphorylation of CDK5RAP2 Ser 140 is necessary for CDK5RAP2-dependent microtubule nucleation. Thus, our findings provide evidence that LRRK1 regulates mitotic spindle orientation downstream of PLK1 through CDK5RAP2-dependent centrosome maturation.