Biology subjects

Rae, J.

Publications and source records attributed to Rae, J..

24 records · Page 2Linked to original sources

bioRxiv2020

An inverted Caveolin-1 topology defines a novel exosome secreted from prostate cancer cells

Caveolin-1 (Cav1) expression and secretion is associated with prostate cancer (PCa) disease progression but the mechanisms underpinning Cav1 release remain poorly understood. Numerous studies have shown Cav1 can be secreted within exosome-like vesicles, but antibody-mediated neutralization can mitigate PCa progression; this is suggestive of an inverted (non-exosomal) Cav1 topology. Here we show that Cav1 can be secreted from specific PCa types in an inverted vesicle-associated form consistent with the features of bioactive Cav1 secretion. Characterization of the isolated vesicles by electron microscopy, single molecule fluorescent microscopy and proteomics reveals they represent a novel class of exosomes [~]40 nm in diameter containing [~]50-60 copies of Cav1 and strikingly, are released via a non-canonical secretory autophagy pathway. This study provides novel insights into a mechanism whereby Cav1 translocates from a normal plasma membrane distribution to an inverted secreted form implicated in PCa disease progression.

cell biology↗

bioRxiv2020

An Endocytic Capture Model for Skeletal Muscle T-tubule Formation

The skeletal muscle T-tubule is a specialized membrane domain essential for coordinated muscle contraction that shows dysmorphology in a number of genetically inherited muscle diseases. However, in the absence of genetically tractable systems the mechanisms involved in T-tubule formation are unknown. Here, we have used the optically transparent and genetically tractable zebrafish system to probe T-tubule development in vivo. By combining live imaging with three-dimensional electron microscopy we derived a four-dimensional quantitative model for T-tubule formation. To elucidate the mechanisms involved in T-tubule formation in vivo we developed a quantitative screen for proteins that associate with and modulate early T-tubule formation including an overexpression screen of the entire zebrafish Rab protein family. We propose a new endocytic capture model involving i) formation of dynamic endocytic tubules at transient nucleation sites on the sarcolemma ii) stabilization by myofibrils/sarcoplasmic reticulum and iii) delivery of membrane from the recycling endosome and Golgi complex.

cell biology↗

bioRxiv2020

Dissecting the nanoscale lipid profile of caveolae

Caveolae are specialized domains of the vertebrate cell surface with a well-defined morphology and crucial roles in cell migration and mechanoprotection. Unique compositions of proteins and lipids determine membrane architectures. The precise caveolar lipid profile and the roles of the major caveolar structural proteins, caveolins and cavins, in selectively sorting lipids have not been defined. Here we used quantitative nanoscale lipid mapping together with molecular dynamic simulations to define the caveolar lipid profile. We show that caveolin1 (CAV1) and cavin1 individually sort distinct plasma membrane lipids. Intact caveolar structures composed of both CAV1 and cavin1 further generate a unique lipid nano-environment. The caveolar lipid sorting capability includes selectivities for lipid headgroups and acyl chains. Because lipid headgroup metabolism and acyl chain remodelling are tightly regulated, this selective lipid sorting may allow caveolae to act as transit hubs to direct communications among lipid metabolism, vesicular trafficking and signalling.

biochemistry↗

bioRxiv2019

Cavin1 intrinsically disordered domains are essential for fuzzy electrostatic interactions and caveola formation

Caveolae are spherically shaped nanodomains of the plasma membrane, generated by cooperative assembly of caveolin and cavin proteins. Cavins are cytosolic peripheral membrane proteins with negatively charged intrinsically disordered regions (DR1-3) that flank positively charged -helical regions (HR1 and HR2). Here we show that the three DR domains of Cavin1 are essential for caveola formation and dynamic trafficking of caveolae. Electrostatic interactions between DR and HR regions promote liquid-liquid phase separation behaviour of Cavin1 in vitro, assembly of Cavin1 oligomers in solution, generation of membrane curvature, association with caveolin-1 (CAV1), and Cavin1 recruitment to caveolae in cells. Removal of the first disordered region causes irreversible gel formation in vitro and results in aberrant caveola trafficking through the endosomal system. We propose a model for caveola assembly whereby fuzzy electrostatic interactions between Cavin1 and CAV1 proteins, combined with membrane lipid interactions, are required to generate membrane curvature and a metastable caveola coat.

cell biology↗

bioRxiv2019

Piezo1 Induces Local Curvature in a Mammalian Membrane and Forms Specific Protein-Lipid Interactions

Touch, hearing, and blood pressure control require mechanically-gated ion channels that convert mechanical stimuli into electrical currents. Piezo1 and Piezo2 were recently identified as essential eukaryotic mechanically-gated ion channels, yet how they respond to physical forces remains poorly understood. Here we use a multi-disciplinary approach to interrogate the interaction of Piezo1 with its lipid environment. We show that individual Piezo1 channels induce significant local curvature in the membrane that is magnified in a cooperative manner to generate larger curved Piezo1 pits. Curvature decreases under lateral membrane tension, consistent with a hypothesis that force detection can involve sensing changes to local curvature. The protein alters its local membrane composition, enriching specific lipids and forming essential binding sites for phosphoinositides and cholesterol that are functionally relevant and often related to Piezo1-mediated pathologies. Finally, we show that Piezo1 alters the expression of lipid-regulating proteins and modifies the cellular lipidome. In short, we find that lipids influence Piezo1 activity and Piezo1 influences the local morphology and composition of the bilayer as well as the cellular lipidome.

biophysics↗

bioRxiv2017

Small GTPases and BAR domain proteins regulate branched actin to make clathrin and dynamin independent endocytic vesicles

Numerous endocytic pathways operate simultaneously at the cell surface. Here we focus on the molecular machinery involved in the generation of endocytic vesicles of the clathrin and dynamin-independent CLIC/GEEC (CG) pathway. This pathway internalises many GPI-anchored proteins and a large fraction of the fluid-phase in different cell types. We developed a real-time TIRF assay using pH-sensitive GFP-GPI to identify nascent CG endocytic sites. The temporal profile of known CG pathway modulators showed that ARF1/GBF1 (GTPase/GEF pair) and CDC42 (RhoGTPase) are recruited sequentially to CG endocytic sites, [~]60s and [~]9s prior to scission. Using a limited RNAi screen, we found several BAR domain proteins affecting CG endocytosis and focused on IRSp53 and PICK1 that have interactions with CDC42 and ARF1 respectively. IRSp53, an I-BAR domain containing protein, was recruited to the plasma membrane at the site of forming CG endocytic vesicles and in its absence, nascent endocytic CLICs, did not form. The requirement for actin polymerization in the CG pathway suggested a role for nucleators of actin polymerization, and ARP2/3 was found enriched at the site of the forming endocytic vesicle. PICK1, a BAR domain containing protein and the ARP2/3 inhibitor is recruited at an early stage along with ARP2/3, but is removed from the endocytic site coincident with CDC42 recruitment and a burst of Factin polymerization. This study provides a spatio-temporal understanding of the molecular machinery necessary to build a CG endocytic vesicle.

cell biology↗