Biology subjects

Itoh, K.

Publications and source records attributed to Itoh, K..

8 recordsLinked to original sources

bioRxiv2022

Onecut1 partially contributes to liver progenitor cell transition and acquisition of metastatic potential in hepatocellular carcinoma.

Metastasis-initiating cells are considered to originate from stem cell-like cancer cells. In hepatocellular carcinoma, liver progenitor-like cells are reported to be derived from hepatocytes, indicating the possible acquisition of metastatic potential during hepatocyte-to-cholangiocyte transdifferentiation. Consistent with the expression pattern observed during ductal plate formation, we revealed an LPC transition with Onecut1 accumulation both during hepatocyte-to-cholangiocyte transdifferentiation and in a cell model. This event may be associated with transient acquisition of metastatic potential.

cancer biology↗

bioRxiv2022

Membrane compression by synaptic vesicle exocytosis triggers ultrafast endocytosis

Compensatory endocytosis keeps the surface area of secretory cells constant following exocytosis. At chemical synapses, clathrin-independent ultrafast endocytosis maintains such homeostasis. This endocytic pathway is temporally and spatially coupled to exocytosis, initiating within 50 ms at the region immediately next to where vesicles fuse: the active zone. How synaptic vesicle exocytosis induces ultrafast endocytosis is unknown. Here, we demonstrate that actin filaments are enriched in the region surrounding active zone at mouse hippocampal synapses and that the membrane area conservation due to this actin corral is necessary for exo-endocytic coupling. Simulations suggest that flattening of fused vesicles exerts lateral membrane pressure in the plasma membrane against the actin corral, resulting in rapid formation of endocytic pits at the border between the active zone and the surrounding actin-enriched region. Consistent with our simulations, ultrafast endocytosis does not initiate when actin organization is disrupted, either pharmacologically or by ablation of the actin-binding protein Epsin1. These data suggest that endocytosis is mechanically coupled to exocytosis at synapses.

cell biology↗

bioRxiv2022

Dynamin forms liquid-like condensates at synapses to support ultrafast endocytosis

Endocytosis at synapses is accelerated by the pre-accumulation of Dynamin 1xA at the endocytic zone by Syndapin 1. However, it is unclear how these proteins support the ultrafast kinetics of endocytosis. Here we report that these proteins phase separate at the presynaptic endocytic zone where ultrafast endocytosis takes place. Specifically, the proline-rich motif of Dynamin 1xA interacts with the Src-Homology 3 domain of Syndapin 1 and forms liquid-like condensates. Single-particle tracking of Dynamin 1xA molecules at synapses shows that their diffusion slows down substantially when they are in the condensates, indicating the presence of molecular crowding and intermolecular interaction. When Dynamin 1xA is mutated to disrupt its interaction with Syndapin 1 the condensates do not form. Thus, the liquid-like assembly of these endocytic proteins provides a catalytic platform for ultrafast endocytosis.

neuroscience↗

bioRxiv2022

Synaptotagmin 7 docks synaptic vesicles for Doc2α-triggered asynchronous neurotransmitter release

Despite decades of intense study, the molecular basis of asynchronous neurotransmitter release remains enigmatic. Synaptotagmin (syt) 7 and Doc2 have both been proposed as Ca2+ sensors that trigger this mode of exocytosis, but conflicting findings have led to controversy. Here, we demonstrate that at excitatory mouse hippocampal synapses, Doc2 is the major Ca2+ sensor for asynchronous release, while syt7 supports this process through activity-dependent docking of synaptic vesicles. In synapses lacking Doc2, asynchronous release after single action potentials is strongly reduced, while deleting syt7 has no effect. However, in the absence of syt7, docked vesicles cannot be replenished on millisecond timescales. Consequently, both synchronous and asynchronous release depress from the second pulse onward during repetitive activity. By contrast, synapses lacking Doc2 have normal activity-dependent docking, but continue to exhibit decreased asynchronous release after multiple stimuli. Moreover, disruption of both Ca2+ sensors is non-additive. These findings result in a new model whereby syt7 drives activity-dependent docking, thus providing synaptic vesicles for synchronous (syt1) and asynchronous (Doc2 and other unidentified sensors) release during ongoing transmission.

neuroscience↗

bioRxiv2021

Defunctionalizing Intracellular Organelles with Genetically-Encoded Molecular Tools Based on Engineered Phospholipase A/Acyltransferases (PLAATs)

Organelles vitally achieve multifaceted functions to maintain cellular homeostasis. Genetic and pharmacological approaches to manipulate individual organelles are powerful in probing their physiological roles. However, many of them are either slow in action, limited to certain organelles, or rely on toxic agents. Here, we designed a generalizable molecular tool utilizing phospholipase A/acyltransferases (PLAATs) for rapid induction of organelle defunctionalization via remodeling of the membrane phospholipid composition. In particular, we identified a minimal, fully catalytic PLAAT with no unfavorable side effects. Chemically-induced translocation of the engineered PLAAT to the mitochondria surface resulted in their rapid deformation in a phospholipase activity dependent manner, followed by loss of luminal proteins as well as dissipated membrane potential, thus invalidating the functionality. To demonstrate wide applicability, we then adapted the molecular tool in peroxisomes, and observed leakage of matrix-resident functional proteins. The technique was compatible with optogenetic control, viral delivery and operation in primary neuronal cultures. Due to such versatility, the PLAAT strategy should present a novel utility in organelle biology of diverse contexts.

cell biology↗

bioRxiv2021

Identification of SSX2IP/Msd1 as a Wtip-binding partner by targeted proximity biotinylation

Wilms tumor-1-interacting protein (Wtip) is a LIM-domain-containing adaptor that links cell junctions with actomyosin complexes and modulates actomyosin contractility and ciliogenesis in Xenopus embryos. The Wtip C-terminus with three LIM domains binds binds Shroom3 and modulates Shroom3-induced apical constriction in ectoderm cells. We found that the N-terminal domain localizes to the basal bodies in skin multiciliated cells, but its interacting partners remain largely unknown. Using a novel targeted proximity biotinylation approach with anti-GFP antibody attached to the biotin ligase BirA in the presence of GFP-Wtip-N, we identified SSX2IP as the candidate binding protein. SSX2IP, also known as Msd1 or ADIP, is a centriolar satellite protein that functions as a targeting factor for ciliary membrane proteins. Wtip physically associated with SSX2IP and the two proteins formed mixed spherical aggregates in overexpressing cells in a dose-dependent manner, in a process that resembles phase separation. These results suggest that the interaction between SSX2IP and Wtip is relevant to their functions at the centrosome and basal bodies. The described antibody targeting of biotin ligase should be applicable to other GFP-tagged proteins.

biochemistry↗

bioRxiv2021

Pinhead antagonizes Admp to promote notochord formation

Dorsoventral patterning of a vertebrate embryo critically depends on the activity of Smad1 that mediates signaling by several BMP proteins, anti-dorsalizing morphogenetic protein (Admp), and their antagonists. Pinhead (Pnhd), a cystine-knot-containing secreted protein, is expressed in the ventrolateral marginal zone during Xenopus gastrulation, however, its molecular targets and signaling mechanisms have not been fully elucidated. An unbiased mass spectrometry-based screen of the gastrula secretome identified Admp as a primary Pnhd-associated protein. We show that Pnhd binds Admp and inhibits its ventralizing activity by reducing Smad1 phosphorylation and suppressing its transcriptional targets. By contrast, Pnhd did not affect the signaling activity of BMP4. Importantly, the Admp gain-of-function phenotype and phospho-Smad1 levels have been enhanced after Pnhd depletion. Furthermore, Pnhd strongly synergized with Chordin and a truncated BMP4 receptor in the induction of notochord markers in ectoderm cells, and Pnhd-depleted embryos displayed notochord defects. Our findings suggest that Pnhd binds and inactivates Admp to promote notochord development. We propose that the interaction between Admp and Pnhd refines Smad1 activity gradients during vertebrate gastrulation.

developmental biology↗

bioRxiv2021

Dynamin is primed at endocytic sites for ultrafast endocytosis

Dynamin mediates fission of vesicles from the plasma membrane during endocytosis. Typically, dynamin is recruited from the cytosol to endocytic sites, requiring seconds to tens of seconds. However, ultrafast endocytosis in neurons internalizes vesicles as quickly as 50 ms during synaptic vesicle recycling. Here we demonstrate that Dynamin 1 is pre-recruited to endocytic sites for ultrafast endocytosis. Specifically, Dynamin 1xA, a splice variant of Dynamin 1, interacts with Syndapin 1 to form molecular condensates on the plasma membrane when the proline-rich domain of this variant is dephosphorylated. When this domain is mutated to include phosphomimetic residues or Syndapin 1s dynamin-interacting domain is mutated, Dynamin 1xA becomes diffuse, and consequently, ultrafast endocytosis slows down by [~]100-fold. Mechanistically, Syndapin 1 acts as an adaptor by binding the plasma membrane and stores Dynamin 1xA at endocytic sites. This cache bypasses the recruitment step and accelerates endocytosis at synapses.

neuroscience↗