1. Since the hallmark of glucose metabolism is insulin-stimulated delivery of glucose transporter-4 (GLUT4) to the plasma membrane (PM) and the hallmark of membrane protein organization is its domain structure, we examined insulins effect on GLUT4 organization in PM of adipose cells. After delivery to PM, all GLUT4 monomers outside domains diffuse freely, but GLUT4 within elongated domains (sized 60-240 nm) diffuse with confinement. Insulin stimulates dissociation of GLUT4 monomers from these domains but does not stimulate monomer-domain association, thereby shifting most PM GLUT4 from clustered to dispersed states. While outside domains, GLUT4 monomers collide frequently but do not form new domains;GLUT4 domain formation is only observed immediately upon exocytosis. Insulin also inhibits exit of GLUT4 from PM, which occurs through endocytosis only at the domains. Thus, insulin not only regulates both exocytosis and endocytosis of GLUT4, it also regulates molecular details of its diffusion, all to control glucose homeostasis. 2. Adipose cells from insulin-resistant human subjects exhibit decreased levels of glucose transporter-4 (GLUT4) and impaired insulin signaling. Here we investigated the dynamics of GLUT4 trafficking and the insulin-stimulated translocation of GLUT4 in adipose cells isolated from human subjects with varying body mass indexes (BMI) and insulin sensitivities (SI). Cells were transfected with HA-GLUT4-GFP/mCherry, and imaged live using total internal reflection fluorescent microscopy to monitor GLUT4 storage vesicle (GSV) trafficking and fusion with the plasma membrane (PM). Confocal microscopy was used to assess the redistribution of HA-GLUT4-GFP to PM using the surface-exposed HA epitope, and to distinguish dispersed from clustered transporters. Without insulin, GSV trafficking on microtubules and fusion with PM, and total cell-surface GLUT4, do not vary with donor subject SI. However, while insulin in cells from insulin-sensitive subjects halts GSV trafficking by stimulating tethering and fusion to PM, and thereby increases cell-surface GLUT4, these effects diminish with decreasing SI, without affecting PM GLUT4 cluster number. In a subgroup of subjects with BMIs of 25 to 35, we found that altered GLUT4 trafficking highly correlated with systemic insulin resistance, independent of BMI. We suggest that development of systemic insulin resistance is associated with maintenance of basal GLUT4 trafficking and PM clusters, but diminished insulin-stimulated GSV tethering and fusion, and cell-surface GLUT4, independent of obesity, and that this altered insulin responsiveness in adipose cells may represent a fundamental mechanistic link between cellular and systemic dysfunction. 3. Biological membrane fission requires protein-driven stress. The GTPase dynamin builds up curvature stress by polymerizing into a helical collar, but the mechanism by which these dynamin collars ensure non-leaky membrane remodeling is actively debated. Using short lipid nanotubes as substrates to directly measure geometric intermediates of the fission pathway, we found that GTP hydrolysis-mediated assembly and disassembly cycles drive dynamin polymerization into short, metastable collars that are optimal for fission. Collars as short as two-rungs can translate radial constriction to reversible hemifission via membrane wedging of the pleckstrin homology domains (PHD) of dynamin. Modeling reveals that tilting of the PHDs to conform with membrane deformations creates the lowest possible energy pathway for hemifission. This local coordination of dynamin and lipids suggests a novel paradigm of membrane remodeling in cells. The theoretical analysis reveals that a stable dynamin polymer constrains highly curved membrane structures thereby effectively inhibiting topological transitions, just as tighter substrate binding inhibits enzymatic catalysis. These constraints are partially relaxed in short and metastable dynamin scaffolds, which not only apply elastic stress (mechano-chemical effects) but also, in coordination with lipids, participate in stochastic searching for the optimal pathway of membrane rearrangements (catalytic effects). The nanoscale coordination between the geometry of the protein scaffold, concerted membrane wedging and the shape of the lipid bilayer results in a distinct geometric catalytic pathway to hemifission. This coordination can be supported by any membrane-inserting protein complex with a ring-like structure, providing a new structural rationale for protein regulatory and catalytic function in membrane remodeling.

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