Mingyue Jin

Rab6a releases LIS1 from a dynein idling complex and activates dynein for retrograde movement

Cytoplasmic dynein drives the movement of a wide range of cargoes towards the minus ends of microtubules. We previously demonstrated that LIS1 forms an idling complex with dynein, which is transported to the plus ends of microtubules by kinesin motors. Here we report that the small GTPase Rab6a is essential for activation of idling dynein. Immunoprecipitation and microtubule pull-down assays reveal that the GTP bound mutant, Rab6a(Q72L), dissociates LIS1 from a LIS1-dynein complex, activating dynein movement in in vitro microtubule gliding assays. We monitor transient interaction between Rab6a(Q72L) and dynein in vivo using dual-colour fluorescence cross-correlation spectroscopy in dorsal root ganglion (DRG) neurons. Finally, we demonstrate that Rab6a(Q72L) mediates LIS1 release from a LIS1-dynein complex followed by dynein activation through an in vitro single-molecule assay using triple-colour quantum dots. Our findings reveal a surprising function for GTP bound Rab6a as an activator of idling dynein.

Katanin p80, NuMA and cytoplasmic dynein cooperate to control microtubule dynamics

Human mutations in KATNB1 (p80) cause severe congenital cortical malformations, which encompass the clinical features of both microcephaly and lissencephaly. Although p80 plays critical roles during brain development, the underlying mechanisms remain predominately unknown. Here, we demonstrate that p80 regulates microtubule (MT) remodeling in combination with NuMA (nuclear mitotic apparatus protein) and cytoplasmic dynein. We show that p80 shuttles between the nucleus and spindle pole in synchrony with the cell cycle. Interestingly, this striking feature is shared with NuMA. Importantly, p80 is essential for aster formation and maintenance in vitro. siRNA-mediated depletion of p80 and/or NuMA induced abnormal mitotic phenotypes in cultured mouse embryonic fibroblasts and aberrant neurogenesis and neuronal migration in the mouse embryonic brain. Importantly, these results were confirmed in p80-mutant harboring patient-derived induced pluripotent stem cells and brain organoids. Taken together, our findings provide valuable insights into the pathogenesis of severe microlissencephaly, in which p80 and NuMA delineate a common pathway for neurogenesis and neuronal migration via MT organization at the centrosome/spindle pole.

Three outer arm dynein heavy chains of Chlamydomonas reinhardtii operate in a coordinated fashion both in vitro and in vivo.

Outer arm dynein (OAD) in cilia and flagella contains two to three nonidentical heavy chains (HCs) that possess motor activity. In Chlamydomonas, flagellar OAD contains three HCs, α‐, β‐, and γ‐HCs, each appearing to have a distinct role. To determine the precise molecular mechanism of their function, cross‐sectional electron micrographs of wild‐type and single HC‐disruption mutants were compared and statistically analyzed. While the α‐HC mutant displayed an OAD of lower density, which was attributed to a lack of α‐HC, the OAD of β‐ and γ‐HC mutants not only lacked the corresponding HC, but was also significantly affected in its structure, particularly with respect to the localization of α‐HC. The lack of β‐HC induced mislocalization of α‐HC, while a disruption of the γ‐HC gene resulted in the synchronized movement of α‐HC and β‐HC in the manners for stacking. Interestingly, using cryo‐electron microscopy, purified OADs were typically observed consisting of two stacked heads and an independent single head, which presumably corresponded to γ‐HC. This conformation is different from previous reports in which the three HCs displayed a stacked form in flagella observed by cryo‐electron tomography and a bouquet structure on mica in deep‐etch replica images. These results suggest that γ‐HC supports the tight stacking arrangement of inter or intra α‐/β‐HC to facilitate the proper functioning of OAD.

Filopodia formation by crosslinking of F‐actin with fascin in two different binding manners

Filopodia are finger‐like protrusions at the leading edge of migrating cells that play a crucial antennal function during cell motility. It is known that actin filaments are bundled hexagonally and provide rigidity to filopodia by virtue of fascin, which plays a central role in actin filament bundling. However, the molecular mechanisms underlying their formation remain unclear. Here, we observed the filopodia of intact whole cells fixed by rapid freezing and revealed their three‐dimensional structure by cryo‐electron tomography and image processing; the actin filament bundling structure by fascin was clarified at high resolution under physiological conditions. It was found that actin filaments in vivo were more numerous than in bundles reconstructed in vitro, and each filopodial actin filament had limited variability in helical twisting. In addition, statistical analysis of actin filament bundles unveiled their detailed architecture. In filopodia, actin filaments had highly ordered structures, and the shift between cross‐links of each adjacent actin filament was approximately 2.7 nm, similar to the monomer repeat of actin filaments. We then proposed a plausible actin‐fascin cross‐link model at the amino acid level and identified three fascin binding sites on two adjacent actin filaments: one filament bound fascin at two discrete, widely separated regions and the other bound fascin in a single small region. We propose that these two different binding modalities should confer rigid bundles that retain flexibility and dynamic performance.

Arl3 and LC8 regulate dissociation of dynactin from dynein

Cytoplasmic dynein acts as a motor for the intracellular retrograde motility of vesicles and organelles along microtubules. However, the regulatory mechanism underlying release of dynactin bound cargoes from dynein motor remains largely unknown. Here we report that ADP-ribosylation factor-like 3 (Arl3) and dynein light chain LC8 induce dissociation of dynactin from dynein. Immunoprecipitation and microtubule pull-down assays revealed that Arl3(Q71L) and LC8 facilitated detachment of dynactin from dynein. We also demonstrated Arl3(Q71L) or LC8-mediated dynactin release from a dynein-dynactin complex through trace experiments using quantum dot (Qdot)-conjugated proteins. Furthermore, we disclosed interactions of Arl3 and LC8 with dynactin and dynein, respectively, by live-cell imaging. Finally, knockdown (KD) of Arl3 and LC8 by siRNA induced abnormal localizations of dynein, dynactin and related organelles. Our findings uncovered the surprising functional relevance of GTP-bound Arl3 and LC8 for the unloading regulation of dynactin-bound cargo from dynein motor.

Alpha-synuclein facilitates to form short unconventional microtubules that have a unique function in the axonal transport

Although α-synuclein (αSyn) has been linked to Parkinson’s disease (PD), the mechanisms underlying the causative role in PD remain unclear. We previously proposed a model for a transportable microtubule (tMT), in which dynein is anchored to a short tMT by LIS1 followed by the kinesin-dependent anterograde transport; however the mechanisms that produce tMTs have not been determined. Our in vitro investigations of microtubule (MT) dynamics revealed that αSyn facilitates the formation of short MTs and preferentially binds to MTs carrying 14 protofilaments (pfs). Live-cell imaging showed that αSyn co-transported with dynein and mobile βIII-tubulin fragments in the anterograde transport. Furthermore, bi-directional axonal transports are severely affected in αSyn and γSyn depleted dorsal root ganglion neurons. SR-PALM analyses further revealed the fibrous co-localization of αSyn, dynein and βIII-tubulin in axons. More importantly, 14-pfs MTs have been found in rat femoral nerve tissue, and they increased approximately 19 fold the control in quantify upon nerve ligation, indicating the unconventional MTs are mobile. Our findings indicate that αSyn facilitates to form short, mobile tMTs that play an important role in the axonal transport. This unexpected and intriguing discovery related to axonal transport provides new insight on the pathogenesis of PD.

Publisher Correction: Alpha-synuclein facilitates to form short unconventional microtubules that have a unique function in the axonal transport

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