Biology subjects

Fukuda, M.

Publications and source records attributed to Fukuda, M..

25 records · Page 2Linked to original sources

bioRxiv2020

Rab34 GTPase mediates ciliary membrane biogenesis in the intracellular ciliogenesis pathway

Primary cilia form by two pathways: an extracellular pathway in which the cilium grows out from the cell surface and an intracellular pathway in which the nascent cilium forms inside the cell. Here we identify the GTPase Rab34 as a selective mediator of intracellular ciliogenesis. We find that Rab34 is required for formation of the ciliary vesicle at the mother centriole and that Rab34 marks the ciliary sheath, a unique sub-domain of assembling intracellular cilia. Rab34 activity is modulated by divergent residues within its GTPase domain, and ciliogenesis requires GTP binding and turnover by Rab34. Because Rab34 is found on assembly intermediates that are unique to intracellular ciliogenesis, we tested its role in the extracellular pathway used by MDCK cells. Consistent with Rab34 acting specifically in the intracellular pathway, MDCK cells ciliate independently of Rab34 and paralog Rab36. Together, these findings reveal a new context-specific molecular requirement for ciliary membrane biogenesis.

cell biology↗

bioRxiv2020

Roles of lysosomotropic agents on LRRK2 activation and Rab10 phosphorylation

Leucine-rich repeat kinase 2 (LRRK2), the major causative gene product of autosomal-dominant Parkinsons disease, is a protein kinase that phosphorylates a subset of Rab GTPases. Since pathogenic LRRK2 mutations increase its ability to phosphorylate Rab GTPases, elucidating the mechanisms of how Rab phosphorylation is regulated by LRRK2 is of great importance. We have previously reported that chloroquine-induced lysosomal stress facilitates LRRK2 phosphorylation of Rab10 to maintain lysosomal homeostasis. Here we reveal that Rab10 phosphorylation by LRRK2 is potently stimulated by treatment of cells with a set of lysosome stressors and clinically used lysosomotropic drugs. These agents commonly promoted the formation of LRRK2-coated enlarged lysosomes and extracellular release of lysosomal enzyme cathepsin B, the latter being dependent on LRRK2 kinase activity. In contrast to the increase in Rab10 phosphorylation, treatment with lysosomotropic drugs did not increase the enzymatic activity of LRRK2, as monitored by its autophosphorylation at Ser1292 residue, but rather enhanced the molecular proximity between LRRK2 and its substrate Rab GTPases on the cytosolic surface of lysosomes. Lysosomotropic drug-induced upregulation of Rab10 phosphorylation was likely a downstream event of Rab29 (Rab7L1)-mediated enzymatic activation of LRRK2. These results suggest a regulated process of Rab10 phosphorylation by LRRK2 that is associated with lysosomal overload stress, and provide insights into the novel strategies to halt the aberrant upregulation of LRRK2 kinase activity.

molecular biology↗

bioRxiv2020

ALIX and ceramide differentially control polarized exosome release from epithelial cells

Exosomes, new players in cell-cell communication, are extracellular vesicles of endocytic origin. Although single cells are known to release various kinds of exosomes (referred to as exosomal heterogeneity), very little is known about the mechanisms by which they are produced and released. Here, we established methods for studying exosomal heterogeneity by using polarized epithelial cells and showed that distinct types of exosomes are differentially secreted from the apical and basolateral sides. We also identified GPRC5C (G protein-coupled receptor class C group 5 member C) as an apical-exosome-specific protein. We further demonstrated that basolateral exosome release depends on ceramide, whereas ALIX, an ESCRT (endosomal sorting complexes required for transport)-related protein, not the ESCRT machinery itself, is required for apical exosome secretion. Thus, two independent machineries, the ALIX-Syntenin1- Syndecan1 machinery (apical side) and the sphingomyelinase-dependent ceramide production machinery (basolateral side), are likely to be responsible for the polarized exosome release from epithelial cells.

cell biology↗

bioRxiv2020

Mechanism of hERG inhibition by gating-modifier toxin, APETx1, deduced by functional characterization

Human ether-a-go-go-related gene potassium channel 1 (hERG) is a voltage-gated potassium channel, the voltage-sensing domain (VSD) of which is targeted by a gating-modifier toxin, APETx1. Although it is known that APETx1 inhibits hERG by stabilizing the resting state, it remains unclear where and how APETx1 interacts with the VSD in the resting state. Here, we prepared a recombinant APETx1, which is structurally and functionally equivalent to the natural product. Electrophysiological analyses using wild type and mutants of APETx1 and hERG revealed that their hydrophobic residues, in addition to a previously reported acidic hERG residue, play key roles in the inhibition of hERG by APETx1. Docking models of the APETx1-VSD complex that satisfy the results of mutational analysis suggest a molecular recognition mode between APETx1 and the resting state of hERG; this would provide a structural basis for designing ligands that control hERG function by binding to the VSD.

molecular biology↗

bioRxiv2020

An Autophagy-Dependent Tubular Lysosomal Network Synchronizes Degradative Activity Required for Muscle Remodeling

Previously, we reported that autophagy is critical for Drosophila muscle remodeling during metamorphosis (Fujita et al., 2017). However, little is known about how lysosomes meet increased degradative demand upon cellular remodeling. Here, we found an extensive tubular autolysosomal network in remodeling muscle. The tubular network transiently appeared and exhibited the capacity to degrade autophagic cargoes. The tubular autolysosomal network was uniquely marked by the autophagic SNARE protein, Syntaxin 17, and its formation depended on both autophagic flux and degradative function, with the exception of the Atg12 and Atg8 ubiquitin-like conjugation systems. Among ATG-deficient mutants, the efficiency of lysosomal tubulation correlated with the phenotypic severity in muscle remodeling. The lumen of the tubular network was continuous and homogeneous across a broad region of the remodeling muscle. Altogether, we revealed that the dynamic expansion of a tubular autolysosomal network synchronizes the abundant degradative activity required for developmentally regulated muscle remodeling. Impact StatementAnalysis of developmentally-regulated Drosophila muscle remodeling revealed autophagy-dependent formation of an extensive, Syntaxin 17-marked, tubular network that synchronizes the abundant degradative activity across a broad region of the remodeling muscle

cell biology↗

bioRxiv2019

Tuba activates Cdc42 during neuronal polarization downstream of the small GTPase Rab8a

The acquisition of neuronal polarity is a complex molecular process that involves several different cellular mechanisms that need to be finely coordinated to define the somatodendritic and axonal compartments. Amongst such mechanisms, cytoskeleton and membrane dynamics control both the morphological transitions that define neuronal polarity acquisition as well as provide molecular determinants to specific sites in neurons at a defined time point. Small GTPases from the Rab and Rho families are well known molecular determinants of neuronal differentiation. However, during axon specification, a molecular link that couples proteins from these two families has yet to be identified. In this paper, we describe the role of Tuba, a Cdc42-specific guanine nucleotide-exchange factor (GEF), in neuronal polarity through a Rab8a-dependent mechanism. Rab8a or Tuba gain-of-function generates neurons with supernumerary axons whereas Rab8a or Tuba loss-of-function abrogated axon specification, phenocopying the well-established effect of Cdc42 on neuronal polarity. Neuronal polarization associated to Rab8a is also evidenced in vivo, since a dominant negative version of Rab8a severely impaired neuronal migration. Remarkably, Rab8a activates Cdc42 in a Tuba-dependent manner, and dominant negative mutants of both GTPases reciprocally prevent the effect over polarity acquisition in the gain-of-function scenarios. Our results strongly suggest that a positive feedback loop linking Rab8a and Cdc42 activities via Tuba, is a primary event in neuronal polarization. In addition, we identified the GEF responsible for Cdc42 activation that is essential to specify axons in cultured neurons.

cell biology↗

bioRxiv2018

Comparable affinity of RabGDIα for GTP- and GDP-bound forms of Rab7 supports a four-state transition model for Rab7 subcellular localization

Endolysosomal system is linked to almost all aspects of cell life and diseases, and Rab7 occupies a critical node in this crucial pathway. However, there have been conflicting views about the exact role of Rab7 in membrane trafficking, since some studies have reported that Rab7 regulates the trafficking from early to late endosomes, while others highlighted its role in late endosomes to lysosomes progression. In the present study, we have revisited this issue from a new viewpoint. In COS-7 cells, a GDP-bound Rab7 mutant, T22N, was located to vesicular membranes as well as in cytoplasm. Similarly, the GTPase-deficient Q67L mutant of Rab7 resided in cytoplasm as well as on membranes. Additionally, we found that RabGDI interacted with both GTP- and GDP-bound forms of Rab7 in vitro. These results have prompted us to propose a four-state transition model for Rab7. This four-state model matches with our recent findings that Rab7 was initially recruited to macropinosomes in a GDP-bound inactive form and subsequently became activated during endocytic maturation in EGF-stimulated COS-7 cells.

cell biology↗