Biology subjects

Kawamata, T.

Publications and source records attributed to Kawamata, T..

5 recordsLinked to original sources

bioRxiv2025

Structural and mechanistic basis for membrane recognition and activation of the vacuolar lipase Atg15

Summary/Abstract Atg15 is a vacuolar phospholipase B essential for the degradation of intravacuolar vesicles such as autophagic bodies. Despite its central role in cellular membrane turnover, the molecular basis of how Atg15 is activated and selectively acts on internal membranes has remained elusive. Here, by combining all-atom and coarse-grained molecular dynamics (MD) simulations with in vitro and in vivo analyses, we elucidate the structural and mechanistic principles underlying Atg15 activation and substrate recognition. Our simulations revealed that disulfide bonds are critical for maintaining the structural integrity of the catalytic core, while the C-terminal region locks the catalytic center in a closed state that prevents activation. Membrane binding induces a transition to an open state, enabling catalysis. Through MD-guided mutational analysis, we identified three regions crucial for catalytic locking, membrane binding, and substrate recognition, and experimentally confirmed that mutations in these regions inhibit activity. Furthermore, Atg15 preferentially associates with positively curved membranes, providing a potential basis for its selective action on internal vesicular membranes. These findings suggest that Atg15s activity is controlled through multiple regulatory layers to ensure safe and selective membrane degradation.

cell biology↗

bioRxiv2023

The mechanism of Atg15-mediated membrane disruption in autophagy

Autophagy is a lysosomal/vacuolar delivery system that isolates and degrades cytoplasmic material. Following delivery by autophagosomes, cytoplasmic components are released into the vacuole within an autophagic body (AB), which is a single-membrane structure derived from the inner membrane of the autophagosome. This membrane must be disrupted for degradation of the cytoplasmic cargo to occur. The vacuolar proteases Pep4 and Prb1, as well as the lipase Atg15, are known to be necessary for this process, but the mechanistic underpinnings remain unclear. In this study, we establish a system to detect lipase activity in the vacuole and use it to show that Atg15 is the sole vacuolar phospholipase and that Pep4 and Prb1 are required for the activation of Atg15 lipase function, which occurs following delivery of Atg15 to the vacuole by the MVB pathway. In vitro experiments also reveal that Atg15 is a B-type phospholipase of broad substrate specificity that is likely implicated in the disruption of a range of membranes delivered to the vacuole. Further, we use isolated ABs to demonstrate that Atg15 alone is able to disrupt AB membranes.

biochemistry↗

bioRxiv2022

Isolation and characterization of autophagic bodies from yeast

Autophagy is a major cellular degradation pathway that is highly conserved among eukaryotes. The identification of cargos captured by autophagosomes is critical to our understanding of the physiological significance of autophagy in cells. In the yeast S. cerevisiae, cells deficient in the vacuolar lipase Atg15 accumulate autophagic bodies (ABs) within the vacuole following the induction of autophagy. As ABs contain cytosolic components including proteins, RNAs, and lipids, their purification allows the identification of material targeted by autophagy for degradation. In this study, we demonstrate a method to purify intact ABs from vacuoles that retain membrane integrity and contain autophagic cargos. This technique offers a valuable tool for the identification of the cargos of autophagy, examination of autophagic cargo selectivity, and biochemical characterization of autophagosome membranes.

molecular biology↗

bioRxiv2019

Encoding of social exploration by neural ensembles in the insular cortex

The insular cortex participates in diverse complex brain functions including sociality, yet little is known about their cellular bases. Using microendoscopic calcium imaging of the agranular insular cortex (AI) in mice interacting with freely-moving and restrained social targets, we identified two subsets of AI neurons -a larger fraction of Social-ON cells and a smaller fraction of Social-OFF cells- that change their activity in opposite directions during social exploration. Social-ON cells included those that represented social investigation independent of location and consisted of multiple subsets, each of which were preferentially active during exploration under particular behavioral state or with a particular target of physical contact. These results uncover a previously unknown function of AI neurons in encoding conjunctive information on social behavior and suggest that AI may act to monitor the ongoing status of social exploration while an animal interacts with unfamiliar conspecifics.

neuroscience↗

bioRxiv2019

Layer-specific sensory processing impairment in the primary somatosensory cortex after motor cortex infarction

Primary motor cortex (M1) infarction occasionally causes sensory impairment. Because sensory signal plays an important role in motor control, sensory impairment compromises recovery and rehabilitation from motor disability. Despite the importance of sensory-motor integration for rehabilitation after M1 infarction, the neural mechanism of the sensory impairment is poorly understood. We show that the sensory processing in the primary somatosensory cortex (S1) was impaired in the acute phase of M1 infarction and recovered in a layer-specific manner in the subacute phase. This layer dependent recovery process and the anatomical connection pattern from M1 to S1 suggested the functional connectivity from M1 to S1 plays a key role in the impairment of sensory processing in S1. The simulation study demonstrated that the loss of inhibition from M1 to S1 in the acute phase of M1 infarction could cause the sensory processing impairment in S1, and the complementation of inhibition could recover the temporal coding. Taken together, we revealed how focal stroke of M1 alters cortical network activity of sensory processing, in which inhibitory input from M1 to S1 may be involved.

physiology↗