Characterisation of GLUT4 trafficking in HeLa cells: Comparable kinetics and orthologous trafficking mechanisms to 3T3-L1 adipocytes

Insulin-stimulated glucose transport is a characteristic property of adipocytes and muscle cells and involves the regulated delivery of glucose transporter (GLUT4)- containing vesicles from intracellular stores to the cell surface. Fusion of these vesicles results in increased numbers of GLUT4 molecules at the cell surface. In an attempt to overcome some of the limitations associated with both primary and cultured adipocytes, we expressed an epitope- and GFP-tagged version of GLUT4 (HA–GLUT4–GFP) in HeLa cells. Here we report the characterisation of this system compared to 3T3-L1 adipocytes. We show that insulin promotes translocation of HA–GLUT4–GFP to the surface of both cell types with similar kinetics using orthologous trafficking machinery. While the magnitude of the insulin-stimulated translocation of GLUT4 is smaller than mouse 3T3-L1 adipocytes, HeLa cells offer a useful, experimentally tractable, human model system. Here, we exemplify their utility through a small-scale siRNA screen to identify GOSR1 and YKT6 as potential novel regulators of GLUT4 trafficking in human cells

Lipid raft microdomain compartmentalization of TC10 is required for insulin signaling and GLUT4 translocation.

Recent studies indicate that insulin stimulation of glucose transporter (GLUT)4 translocation requires at least two distinct insulin receptor-mediated signals: one leading to the activation of phosphatidylinositol 3 (PI-3) kinase and the other to the activation of the small GTP binding protein TC10. We now demonstrate that TC10 is processed through the secretory membrane trafficking system and localizes to caveolin-enriched lipid raft microdomains. Although insulin activated the wild-type TC10 protein and a TC10/H-Ras chimera that were targeted to lipid raft microdomains, it was unable to activate a TC10/K-Ras chimera that was directed to the nonlipid raft domains. Similarly, only the lipid raft-localized TC10/ H-Ras chimera inhibited GLUT4 translocation, whereas the TC10/K-Ras chimera showed no significant inhibitory activity. Furthermore, disruption of lipid raft microdomains by expression of a dominant-interfering caveolin 3 mutant (Cav3/DGV) inhibited the insulin stimulation of GLUT4 translocation and TC10 lipid raft localization and activation without affecting PI-3 kinase signaling. These data demonstrate that the insulin stimulation of GLUT4 translocation in adipocytes requires the spatial separation and distinct compartmentalization of the PI-3 kinase and TC10 signaling pathways

Dynamics of the plasma membrane transporter GLUT4

Glucose homeostasis in the human body is maintained by hormones of the pancreas, mostly glucagon and insulin. Insulin is secreted when blood glucose levels are high and triggers a signalling cascade that results in glucose uptake via the glucose transporter GLUT4 in peripheral tissues. GLUT4 is the only glucose transporter that responds to insulin stimulation and it slowly recycles between intracellular storage compartments and the plasma membrane. In the basal state, the majority of GLUT4 is intracellularly localised. Insulin stimulation results in movement (“translocation”) of GLUT4 from these intracellular stores to the plasma membrane. The signalling cascade from insulin binding to its receptor to translocation of GLUT4 is comparatively well understood. Less is known about the dynamics of GLUT4 within the plasma membrane itself. Advances in light microscopy techniques, such as Total Internal Reflection Fluorescence and super-resolution microscopy, have allowed new insights into the events in the membrane. It has recently been proposed that GLUT4 is located in plasma membrane clusters and that another effect of insulin is the dispersal of these GLUT4 clusters. The main objective of this work was to develop a microscopy-based assay to visualise and quantify these clusters and to investigate the molecular mechanisms behind clustering and dispersal of the glucose transporter in response to insulin. The majority of this work has been carried out in 3T3 L1 adipocytes, a widely used cell model for the study of GLUT4. However, this cell line is difficult to maintain, and its genetic manipulation is very challenging. For this reason, we investigated HeLa cells as a suitable substitute cell model for preliminary screenings. Using Total Internal Reflection Fluorescence Microscopy and Spatial Intensity Distribution Analysis, we gained new insight into the dynamics of plasma membrane GLUT4 in both 3T3 L1 adipocytes and HeLa cells. We found that the transporter forms an oligomer of high order in the plasma membrane in both cell types. Further, we compared the dynamics of GLUT4 mobilisation in response to insulin and found similar results. Based on these findings, we carried out an siRNA knock-down screening to determine proteins involved in intracellular GLUT4 trafficking and found that GOSR1 and Ykt6 are promising targets for further examination. Single molecule localisation microscopy allowed us to accomplish our aim to assay GLUT4 clustering and dispersal. Using dSTORM and Ripley’s K-function, as well as Bayesian cluster analysis methods, we showed that GLUT4 is indeed located in clusters in the plasma membrane and that insulin stimulation leads to its dispersal. We found that treatment with Galectin-3, a drug that inhibits glucose uptake, impedes the dispersal. Building upon previous research in our group that identified EFR3a as a membrane-localised protein involved in glucose uptake, we knocked-down EFR3a in 3T3 L1 adipocytes and found that this also disrupts GLUT4 dispersal, which we hypothesise could be a potential drug target for type 2 diabetes. Taken together, the findings presented in this thesis suggest HeLa cells as a suitable cell model for initial assessments of research questions related to GLUT4 trafficking. Furthermore, a robust assay to measure GLUT4 dispersal was established

Fish and mammalian glut4 traffic characteristics: an evolutionary perspective on the importance of glut4 protein motifs for trafficking

[eng] Glucose transporters (GLUTs) are extremely important for glucose metabolism. Glucose transporters uptake glucose from blood stream into the cells where it can be metabolized. Among the glucose transporters family, GLUT4, which is solely expressed in muscle and adipose tissues, displays a unique feature as it can change its cellular distribution within minutes in response to insulin to regulate glucose uptake. Therefore, the study of GLUT4 cellular trafficking is fundamental to understand its functioning and to deepen our knowledge on glucose homeostasis. In this work, we utilized a GLUT4 fish variant, brown trout GLUT4, to study GLUT4 trafficking and the role of GLUT4 protein motifs in this process, in 3T3-L1 adipocytes. We observed that, in comparison to mammalian GLUT4 (RatGLUT4), brown trout GLUT4 (BtGLUT4) had a much weaker translocation to the plasma membrane in response to insulin which was in part due to a slower cellular trafficking (exocytosis and endocytosis) and to a poor targeting to the GLUT4 storage vesicles responsible for “holding” GLUT4 inside the cell in the absence of insulin; these vesicles represent the main pool of insulin-responsive GLUT4. In this thesis we also studied the most common GLUT4 endocytic routes. We analyzed the contribution of the clathrin-mediated and the cholesterol-dependent endocytic pathways for RatGLUT4 and BtGLUT4 internalization. We observed that whilst RatGLUT4 internalizes through both the clathrin-mediated and the cholesterol-dependent pathways, BtGLUT4 only utilizes the former. It has been suggested that in adipocytes, the main cholesterol-dependent internalization pathway is the caveolar route. The internalization through this pathway is mediated by plasma membrane structures called caveolae. The formation of these structures is dependent on the caveolin-1 protein. To analyze the role of caveolae in GLUT4 internalization we blocked its formation by knocking down caveolin-1 and observed an increase of RatGLUT4 and BtGLUT4 internalization; however, both GLUT4 isoforms showed less internalization through the clathrin-mediated and cholesterol-dependent pathways in the absence of cavolin-1. Therefore, we suggest that in 3T3-L1 adipocytes caveolin-1 knockdown induces internalization of GLUT4 through alternative pathways. GLUT4 trafficking is regulated by cellular machinery that interacts with GLUT4 protein motifs. To analyze the role of the mammalian N-terminal FQQI8 and C-terminal TELEY502 motifs in GLUT4 trafficking we mutated the corresponding motifs in BtGLUT4 (FQHL8 and TELDY495, respectively) and observed that mutations in the C-terminal had little effect on BtGLUT4 trafficking whereas mutations on the N-terminal (especially FQQL8 mutant) improved BtGLUT4 intracellular retention in the absence of insulin. Furthermore, we verified that FQQL8 mutation increased BtGLUT4 retention in a syntaxin-6-rich compartment, possibly the trans-Golgi network. In addition to studying BtGLUT4 mutants we also analyzed the trafficking of a chimera consisting of a RatGLUT4 backbone with the large cytoplasmic loop of BtGLUT4 (L-GLUT4). We observed that L-GLUT4 possessed higher plasma membrane levels in the absence of insulin and as a result a weaker translocation. Moreover, we observed that this was caused, at least in part, by a reduction in the endocytosis of L-GLUT4 in the absence of insulin. We also analyzed the contribution of the clathrin-mediated and cholesterol-dependent pathways for L-GLUT4 internalization and observed that the loop substitution (L-GLUT4) reduced RatGLUT4 internalization through the cholesterol-dependent route. Moreover, in the absence of insulin and in caveolin-1, L-GLUT4 internalization did not increase as much as that of RatGLUT4. The internalization of L-GLUT4 in the absence of caveolin-1 and insulin occurred through a clathrin-mediated pathway, similarly to BtGLUT4, but it also internalized through a cholesterol-dependent pathway, unlike RatGLUT4 and BtGLUT4. In summary, in this thesis we have contributed to increase the knowledge on GLUT4 trafficking and on the roles of the FQQI8 motif and large cytoplasmic loop in this process, in 3T3-L1 adipocytes.[spa] El transportador de glucosa GLUT4 tiene la capacidad de, en respuesta a insulina, cambiar su localización celular y de esta forma regular el transporte de glucosa. En este trabajo, hemos utilizado una variante de GLUT4 de trucha (BtGLUT4) para estudiar el trafico de GLUT4, así como sus dominios proteicos involucrados en este proceso, en adipocitos 3T3-L1. Hemos observado que en comparación con el GLUT4 de mamíferos (RatGLUT4), el BtGLUT4 tenia una menor capacidad de translocación a la membrana plasmática en respuesta a insulina y que esto se debía a una trafico celular mas lento (exocitosis y endocitosis) y a una peor retención en las vesículas responsables por retener el transportador dentro de la célula en ausencia de insulina. En este trabajo hemos observado que RatGLUT4 ha internalizado por la vía de endocitosis mediada por clatrina y por la vía dependiente de colesterol, mientras que BtGLUT4 solo ha utilizado la primera. Además, hemos inhibido la internalización caveolar, mediante bajada de la expresión de caveolina-1, y hemos observado un aumento de la internalización de RatGLUT4 y BtGLUT4. Con el objetivo de estudiar el papel del dominio FQQI8 (extremo -N) de mamífero en el trafico de GLUT4, hemos mutado la secuencia correspondiente en BtGLUT4 (FQHL8) y hemos observado que mutaciones en este dominio han mejorado la retención intracelular de BtGLUT4 en ausencia de insulina. También hemos estudiado el trafico de una quimera que consiste en la secuencia de RatGLUT4 con el lazo citoplasmático largo de BtGLUT4 (L-GLUT4). Hemos observado que la sustitución del lazo ha aumentado los niveles de RatGLUT4 en superficie en ausencia de insulina y que esto era debido, por lo menos en parte, a una menor endocitosis en ausencia de la hormona. También hemos observado que la sustitución del lazo de RatGLUT4 ha reducido su internalización a través de la vía dependiente de colesterol en ausencia de insulina. Además, en ausencia de caveolina-1 y insulina, la internalización de L-GLUT4 ha aumentado menos que la de RatGLUT4 y ha ocurrido a través de las vías mediada por clatrina y dependiente de colesterol

Investigating the Role of Microtubules in Glut4 Vesicle Trafficking and the Kinetics of Membrane Attachment by the Myosin Myo1c

The myosin myo1c dynamically localizes to cellular membranes through high affinity phosphoinositide binding and links them to the actin cytoskeleton. Determining the kinetics of membrane attachment will provide insight into the relationship between membrane-attachment and actin-attachment lifetimes, and will also provide details about the regulation of membrane attachment. Stopped-flow spectroscopy was used to measure the binding and dissociation of a recombinant myo1c construct containing the tail and regulatory domains (myo1cIQ-tail) to and from 100 nm diameter large unilamellar vesicles (LUVs). The apparent second-order rate constant for association of myo1cIQ-tail with LUVs containing 2% phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) was approximately diffusion-limited. Myo1cIQ-tail dissociated from PtdIns(4,5)P2 at a slower rate (2.0 s-1) than the pleckstrin homology domain of phospholipase C-δ (PLCδ-PH) (13 s-1). The presence of additional anionic phospholipid reduced the myo1cIQ-tail dissociation rate constant \u3e 50-fold, but marginally changed the dissociation rate of PLCδ-PH, suggesting that additional electrostatic interactions in myo1cIQ-tail help to stabilize binding. Remarkably, high concentrations of soluble inositol phosphates induce dissociation of myo1cIQ-tail from LUVs, suggesting that phosphoinositides are able to bind and dissociate from myo1cIQ-tail as it remains bound to the membrane. In adipocytes, vesicles containing glucose transporter-4 (GLUT4) redistribute from intracellular stores to the cell periphery in response to insulin. Vesicles then fuse with the plasma membrane, facilitating glucose transport into the cell. To gain insight into the molecular role of microtubules, we examined the spatial organization and dynamics of microtubules in relation to GLUT4 vesicle trafficking in living 3T3-L1 adipocytes using total internal reflection fluorescence (TIRF) microscopy. Insulin stimulated an increase in microtubule density and curvature within the TIRF-illuminated region of the cell. The time course of the density increase precedes that of the increase in intensity of HA-GLUT4-eGFP in this same region. Microtubule disruption delayed and modestly reduced the accumulation of GLUT4 at the plasma membrane. Interestingly, fusion of GLUT4-containing vesicles with the plasma membrane preferentially occur near microtubules, and long-distance vesicle movement along microtubules visible at the cell surface prior to fusion does not appear to account for this proximity. We conclude that microtubules may be important in providing spatial information for fusion events

A Deep Learning Framework for Automated Vesicle Fusion Detection

Quantitative analysis of vesicle-plasma membrane fusion events in the fluorescence microscopy, has been proven to be important in the vesicle exocytosis study. In this paper, we present a framework to automatically detect fusion events. First, an iterative searching algorithm is developed to extract image patch sequences containing potential events. Then, we propose an event image to integrate the critical image patches of a candidate event into a single-image joint representation as the input to Convolutional Neural Networks (CNNs). According to the duration of candidate events, we design three CNN architectures to automatically learn features for the fusion event classification. Compared on 9 challenging datasets, our proposed method showed very competitive performance and outperformed two state-of-the-arts

Quantitative immunofluorescence microscopy of subcellular GLUT4 distribution in human skeletal muscle: effects of endurance and sprint interval training.

Increases in insulin-mediated glucose uptake following endurance training (ET) and sprint interval training (SIT) have in part been attributed to concomitant increases in glucose transporter 4 (GLUT4) protein content in skeletal muscle. This study used an immunofluorescence microscopy method to investigate changes in subcellular GLUT4 distribution and content following ET and SIT. Percutaneous muscle biopsy samples were taken from the m. vastus lateralis of 16 sedentary males in the overnight fasted state before and after 6 weeks of ET and SIT. An antibody was fully validated and used to show large (> 1 μm) and smaller (<1 μm) GLUT4-containing clusters. The large clusters likely represent trans-Golgi network stores and the smaller clusters endosomal stores and GLUT4 storage vesicles (GSVs). Density of GLUT4 clusters was higher at the fibre periphery especially in perinuclear regions. A less dense punctate distribution was seen in the rest of the muscle fibre. Total GLUT4 fluorescence intensity increased in type I and type II fibres following both ET and SIT. Large GLUT4 clusters increased in number and size in both type I and type II fibres, while the smaller clusters increased in size. The greatest increases in GLUT4 fluorescence intensity occurred within the 1 μm layer immediately adjacent to the PM. The increase in peripheral localisation and protein content of GLUT4 following ET and SIT is likely to contribute to the improvements in glucose homeostasis observed after both training modes

GLUT4 dispersal at the plasma membrane of adipocytes : a super-resolved journey

In adipose tissue, insulin stimulates glucose uptake by mediating the translocation of GLUT4 from intracellular vesicles to the plasma membrane. In 2010, insulin was revealed to also have a fundamental impact on the spatial distribution of GLUT4 within the plasma membrane, with the existence of two GLUT4 populations at the plasma membrane being defined: 1) as stationary clusters and 2) as diffusible monomers. In this model, in the absence of insulin, plasma membrane-fused GLUT4 are found to behave as clusters. These clusters are thought to arise from an exocytic event that retains GLUT4 at the fusion site; this has been proposed to function as an intermediate hub between GLUT4 exocytosis and re-internalisation. By contrast, insulin stimulation induces the dispersal of GLUT4 clusters into monomers and favors a distinct type of GLUT4-vesicle fusion event, known as fusion-with-release exocytosis. Here, we review how super-resolution microscopy approaches have allowed investigation of the characteristics of plasma membrane-fused GLUT4 and further discuss regulatory step(s) involved in the GLUT4 dispersal machinery, introducing the scaffold protein EFR3 which facilitates localisation of phosphatidylinositol 4-kinase type IIIα (PI4KIIIα) to the cell surface. We consider how dispersal may be linked to the control of transporter activity, consider whether macro-organisation may be a widely used phenomenon to control proteins within the plasma membrane, and speculate on the origin of different forms of GLUT4-vesicle exocytosis. [Abstract copyright: Copyright 2023 The Author(s).

The role of mVps45 in regulating GLUT4 trafficking in 3T3L1 adipocytes

Insulin stimulates glucose transport in fat cells by inducing the movement of glucose transporters (Glucose transporter-4) from specialised storage vesicles to the plasma membrane. Insulin resistant individuals and those with Type II Diabetes exhibit impairment in the ability of insulin to stimulate glucose transport. The molecular mechanisms of glucose transporter-4 trafficking in adipocytes are an important focus in understanding the underlying etiology of this disease. Glucose transporter-4 (GLUT4) recycles between the plasma membrane and intracellular stores in the absence of insulin using a complex intracellular pathway. This involves two intracellular cycles: one is the prototypical endosomal system, the other a specialised cycle involving the trans-Golgi network and a sub-set of intracellular vesicles called GSVs (the slow cycle). Understanding the control of the entry into this second cycle is the subject of this thesis. In particular, the work in this thesis will examine the role of Syntaxin 16 and its cognate Sec1/Munc18 protein mammalian Vps45 (mVps45). The regulation of Syntaxin 16 has not been fully elucidated and understanding the role of Syntaxin 16 in SNARE complex regulation and subsequent control of GLUT4 traffic into the slow cycle requires an understanding of its cognate binding partner Sec1/Munc18 (SM) protein, mammalian Vps45 (mVps45). The absolute levels of both Syntaxin 16 and mVps45 were quantified and found to be present in 3T3-L1 adipocytes in roughly stoichiomeric amounts. IP experiments also showed the ability of mVps45 to interact with Syntaxin 16 in the absence of insulin. Using the model eukaryote Saccharomyces cerevisiae, we found that mVps45 could complement for the deletion of Vps45p. Assays for CPY secretion showed that mVps45 is able to complement for the loss of Vps45p function in the trafficking of carboxypeptidase Y (CPY). Additionally, mVps45 mutants were made that correspond to yeast mutants made previously in the lab and were tested for homology of function. Depleting 3T3-L1 adipocytes of mVps45 showed alterations in the levels of GLUT4 protein as well as the protein levels of Syntaxin 16, IRAP, and Rabenosyn. Insulin-stimulated deoxyglucose uptake was also profoundly decreased upon depletion of mVps45. Further experiments using mVps45 depleted cells show that these cells lose their sensitivity to insulin and that the loss of mVps45 in these cells causes GLUT4 to have the inability to enter the slow cycle. Taken together, these findings demonstrate that mVps45 has a role in allowing GLUT4 entry into the slow cycle