Biology subjects

Larocque, G.

Publications and source records attributed to Larocque, G..

6 recordsLinked to original sources

bioRxiv2026

The GLUT4 storage vesicle pool is maintained by intracellular nanovesicle traffic

Glucose transporter 4 (GLUT4) is sequestered intracellularly until the action of insulin causes its redistribution to the cell surface. How cells sequester GLUT4, generating and maintaining GLUT4 storage vesicles (GSVs), is still unclear. Intracellular nanovesicles (INVs) operate on various membrane trafficking pathways and show molecular similarity to GSVs. Here we show that GLUT4 and associated GSV cargo proteins (IRAP/LNPEP, cellugyrin/SYNGR2, sortilin/SORT1, and VAMP2) are trafficked by INVs, which we term GLUT4-flavor INVs. The majority of GLUT4 is carried in GLUT4-flavor INVs which represent a small fraction of the total INV pool. We use a method to capture specific vesicles and test their involvement in the redistribution of GLUT4 in response to insulin. Direct capture of defined GSVs ablated the response, however capture of INVs had a smaller effect. We show that the function of GLUT4-flavor INVs is in the sorting of GSV proteins to supply the insulin-responsive GSV pool and that interfering with INV function by TPD54/TPD52L2 depletion results in mistrafficking of GLUT4 to the plasma membrane. These findings establish GLUT4-flavor INVs as the precursor GSVs that are responsible for intracellular sequestration of GLUT4.

cell biology↗

bioRxiv2022

Recruitment of clathrin to intracellular membranes is sufficient for vesicle formation

The formation of a clathrin-coated vesicle is a major membrane remodeling process that is crucial for membrane traffic in cells. Besides clathrin, these vesicles contain at least 100 different proteins although it is unclear how many are essential for the formation of the vesicle. Here, we show that intracellular clathrin-coated formation can be induced in living cells using minimal machinery and that it can be achieved on various membranes, including the mitochondrial outer membrane. Chemical heterodimerization was used to inducibly attach a clathrin-binding fragment "hook" to an "anchor" protein targeted to a specific membrane. Endogenous clathrin assembled to form coated pits on the mitochondria, termed MitoPits, within seconds of induction. MitoPits are double-membraned invaginations that form preferentially on high curvature regions of the mitochondrion. Upon induction, all stages of CCV formation - initiation, invagination, and even fission - were faithfully reconstituted. We found no evidence for the functional involvement of accessory proteins in this process. In addition, fission of MitoPit-derived vesicles was independent of known scission factors including dynamins and dynamin-related protein 1 (Drp1), suggesting that the clathrin cage generates sufficient force to bud intracellular vesicles. Our results suggest that, following its recruitment, clathrin alone is sufficient for intracellular clathrin-coated vesicle formation.

cell biology↗

bioRxiv2021

Multi-modal adaptor-clathrin contacts drive coated vesicleassembly

Clathrin-coated pits are formed by the recognition of membrane and cargo by the heterotetrameric AP2 complex and the subsequent recruitment of clathrin triskelia. A potential role for AP2 in coated-pit assembly beyond initial clathrin recruitment has not been explored. Clathrin binds the {beta}2 subunit of AP2, and several binding sites on {beta}2 and on the clathrin heavy chain have been identified, but our structural knowledge of these interactions is incomplete and their functional importance during endocytosis is unclear. Here, we analysed the cryo-EM structure of clathrin cages assembled in the presence of {beta}2 hinge and appendage ({beta}2HA) domains. We find that the {beta}2-appendage binds in at least two positions in the cage, demonstrating that multi-modal binding is a fundamental property of clathrin-AP2 interactions. In one position, {beta}2-appendage cross-links two adjacent terminal domains from different triskelia below the vertex. Functional analysis of {beta}2HA-clathrin interactions reveals that endocytosis requires two clathrin interaction sites: a clathrin-box motif on the hinge and the "sandwich site" on the appendage, with the appendage "platform site" having less importance. From these studies and the work of others, we propose that {beta}2-appendage binding to more than one clathrin triskelion is a key feature of the system and likely explains why clathrin assembly is driven by AP2.

biochemistry↗

bioRxiv2020

Intracellular nanovesicles mediate integrin trafficking during cell migration

Membrane traffic is an important regulator of cell migration through the endocytosis and recycling of cell surface receptors such as integrin heterodimers. Intracellular nanovesicles (INVs), are a recently identified class of transport vesicle that are involved in multiple membrane trafficking steps including the recycling pathway. The only known marker for INVs is Tumor Protein D54 (TPD54/TPD52L2), a member of the TPD52-like protein family. Overexpression of TPD52-like family proteins in cancer has been linked to poor prognosis and an aggressive metastatic phenotype which suggests cell migration may be altered under these conditions. Here we show that TPD54 associates with INVs by directly binding high curvature membrane via a conserved positively charged motif in its C-terminus. We describe how other members of the TPD52-like family are also associated with INVs and we document the Rab GTPase complement of all INVs. Depletion of TPD52-like proteins inhibits cell migration and invasion; and we show that this is likely due to altered integrin recycling. Our study highlights the involvement of INVs in the trafficking of cell surface proteins to generate biologically important outputs in health and disease.

cell biology↗

bioRxiv2019

Unintended inhibition of protein function using GFP nanobodies in human cells

Tagging a protein-of-interest with GFP using genome editing is a popular approach to study protein function in cell and developmental biology. To avoid re-engineering cell lines or organisms in order to introduce additional tags, functionalized nanobodies that bind GFP can be used to extend the functionality of the GFP tag. We developed functionalized nanobodies, which we termed \"dongles\", that could add, for example, an FKBP tag to a GFP-tagged protein-of-interest; enabling knocksideways experiments in GFP knock-in cell lines. The power of knocksideways is that it allows investigators to rapidly switch the protein from an active to an inactive state. We show that dongles allow for effective knocksideways of GFP-tagged proteins in genome-edited human cells. However, we discovered that nanobody binding to dynamin-2-GFP caused inhibition of dynamin function prior to knocksideways. While this limitation might be specific to the protein studied, it was significant enough to convince us not to pursue development of dongle technology further.

cell biology↗

bioRxiv2018

Tumor Protein D54 is a promiscuous Rab effector

Transport of proteins and lipids from one membrane compartment to another is via intracellular vesicles. We investigated the function of Tumor Protein D54 (TPD54/TPD52L2), and found that TPD54 was involved in multiple membrane trafficking pathways: anterograde traffic, recycling and Golgi integrity. To understand how TPD54 controls these diverse functions, we used an inducible method to reroute TPD54 to mitochondria. Surprisingly, this manipulation resulted in the capture of many small vesicles (30 nm diameter) at the mitochondrial surface. Super-resolution imaging confirmed the presence of similarly sized TPD54-positive structures under normal conditions. It appears that TPD54 defines a new class of transport vesicle, which we term intracellular nanovesicles (INVs). INVs meet three criteria for functionality. They contain specific cargo, they have certain R-SNAREs for fusion, and they are endowed with a variety of Rab GTPases (16 out of 43 tested). The molecular heterogeneity of INVs and the diverse functions of TPD54 suggest that INVs have various membrane origins and a number of destinations. We propose that INVs are a generic class of transport vesicle which transfer cargo between these varied locations.

cell biology↗