Date of Award

2012

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

E. M. Ostap

Abstract

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 > 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.

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