Biology subjects

Konno, T.

Publications and source records attributed to Konno, T..

4 recordsLinked to original sources

bioRxiv2022

ERnet: a tool for the semantic segmentation and quantitative analysis of endoplasmic reticulum topology for video-rate super-resolution imaging

The topology of endoplasmic reticulum (ER) network is highly regulated by various cellular and environmental stimuli and affects major functions such as protein quality control and the cells response to metabolic changes. The ability to quantify the dynamical changes of the ER structures in response to cellular perturbations is crucial for the development of novel therapeutic approaches against ER associated diseases, such as hereditary spastic paraplegias and Niemann Pick Disease type C. However, the rapid movement and small spatial dimension of ER networks make this task challenging. Here, we combine video-rate super-resolution imaging with a state-of-the-art semantic segmentation method capable of automatically classifying sheet and tubular ER domains inside individual cells. Data are skeletonised and represented by connectivity graphs to enable the precise and efficient quantification and comparison of the network connectivity from different complex ER phenotypes. The method, called ERnet, is powered by a Vision Transformer architecture, and integrates multi-head self-attention and channel attention into the model for adaptive weighting of frames in the time domain. We validated the performance of ERnet by measuring different ER morphology changes in response to genetic or metabolic manipulations. Finally, as a means to test the applicability and versatility of ERnet, we showed that ERnet can be applied to images from different cell types and also taken from different imaging setups. Our method can be deployed in an automatic, high-throughput, and unbiased fashion to identify subtle changes in cellular phenotypes that can be used as potential diagnostics for propensity to ER mediated disease, for disease progression, and for response to therapy.

bioengineering↗

bioRxiv2022

Intracellular Aβ42 aggregation leads to cellular thermogenesis

The aggregation of A{beta}42 is a hallmark of Alzheimers disease. It is still not known what the biochemical changes are inside a cell which will eventually lead to A{beta}42 aggregation. Thermogenesis has been associated with cellular stress, the latter of which may promote aggregation. We perform intracellular thermometry measurements using fluorescent polymeric thermometers (FPTs) to show that A{beta}42 aggregation in live cells leads to an increase in cell-averaged temperatures. This rise in temperature is mitigated upon treatment with an aggregation inhibitor of A{beta}42 and is independent of mitochondrial damage that can otherwise lead to thermogenesis. With this, we present a diagnostic assay which could be used to screen small-molecule inhibitors to amyloid proteins in physiologically relevant settings. To interpret our experimental observations and motivate the development of future models, we perform classical molecular dynamics of model A{beta} peptides to examine the factors that hinder thermal disspation. We observe that this is controlled by the presence of ions in its surrounding environment, the morphology of the amyloid peptides and the extent of its hydrogen-bonding interactions with water. We show that aggregation and heat retention by A{beta} peptides are favoured under intracellular-mimicking ionic conditions, which could potentially promote thermogenesis. The latter will, in turn, trigger further nucleation events that accelerate disease progression.

biophysics↗

bioRxiv2021

Endoplasmic Reticulum morphological regulation by RTN4/NOGO modulates neuronal regeneration by slowing luminal transport

Cell and tissue functions rely on an elaborate intracellular transport system responsible for distributing bioactive molecules with high spatiotemporal accuracy. The tubular network of the Endoplasmic Reticulum (ER) constitutes a system for the delivery of luminal solutes it stores, including Ca2+, across the cell periphery. The physical nature and factors underlying the ERs functioning as a fluidics system are unclear. Using an improved ER transport visualisation methodology combined with optogenetic Ca2+ dynamics imaging, we observed that ER luminal transport is modulated by natural ER tubule narrowing and dilation, directly proportional to the amount of an ER membrane morphogen, Reticulon 4 (RTN4). Consequently, the ER morphoregulatory effect of RTN4 defines ERs capacity for peripheral Ca2+ delivery and thus controls axonogenesis. Excess RTN4 limited ER luminal transport, Ca2+ release and iPSC-derived cortical neurons axonal extension, while RTN4 elimination reversed the effects. SummaryIntracellular transport through the lumen of the ER network is modulated through narrowing/dilation of ER tubules by a membrane morphogen - RTN4, a process controlling axonogenesis by limiting the delivery of ER-stored Ca2+.

cell biology↗

bioRxiv2021

Stress-induced protein disaggregation in the Endoplasmic Reticulum catalysed by BiP

Protein synthesis is supported by cellular machineries that ensure polypeptides fold to their native three-dimensional conformation with high fidelity whilst eliminating misfolded, aggregation-prone species. While protein aggregates can contribute to pathologies exemplified by Alzheimers and Parkinsons diseases, their abundance is normally minimised by molecular chaperones such as HSP70/90, which promote native folding and drive the recycling of aberrantly folded species. Cytoplasmic chaperones (e.g. HSP70/40) can resolve insoluble protein aggregates should the preventive mechanisms falter. However, it is unknown whether an analogous disaggregation system is needed and exists in the Endoplasmic Reticulum (ER), where [~]30% of the proteome is synthesised. Here we show that the ER of a variety of mammalian cell types, including neurons, is endowed with the capability to resolve protein aggregates that accumulate upon expression of metastable proteins. Utilising a purpose-developed protein aggregation probing system with a sub-organellar resolution, we observed steady-state aggregate accumulation in the ER. Strikingly, pharmacological induction of ER stress did not augment aggregates but rather stimulated their clearance within hours. We found that this disaggregation activity was catalysed by the stress-responsive ER molecular chaperone - BiP. Its elimination abolished the disaggregation activity in cells. Further, we reconstructed the disaggregation-reaction in-vitro by a minimal system of ATP-fuelled BiP and its J-protein cofactor. These data reveal a hitherto unknown, non-redundant function of the ER stress response. Thus, our findings may facilitate the identification of aggregation-antagonising strategies and rationalising the age-dependent protein misfolding pathology. SummaryER stress induction activates a protein disaggregation machinery, powered by BiP - an abundant ER chaperone, revealed by a FLIM-based protein aggregation monitoring in live cells.

molecular biology↗