In vitro gentamicin exposure alters caveolae protein profile in cochlear spiral ligament pericytes.

BACKGROUND: The aminoglycoside antibiotic gentamicin is an ototoxic drug and has been used experimentally to investigate cochlear damage induced by noise.We have investigated the changes in the protein profile associated with caveolae in gentamicin treated and untreated spiral ligament (SL) pericytes, specialized cells in the blood labyrinth barrier of the inner ear microvasculature. Pericytes from various microvascular beds express caveolae, protein and cholesterol rich microdomains, which can undergo endocytosis and transcytosis to transport small molecules in and out the cells. A different protein profile in transport-specialized caveolae may induce pathological changes affecting the integrity of the blood labyrinth barrier and ultimately contributing to hearing loss. METHOD: Caveolae isolation from treated and untreated cells is achieved through ultracentrifugation of the lysates in discontinuous gradients. Mass spectrometry (LC-MS/MS) analysis identifies the proteins in the two groups. Proteins segregating with caveolae isolated from untreated SL pericytes are then compared to caveolae isolated from SL pericytes treated with the gentamicin for 24 h. Data are analyzed using bioinformatic tools. RESULTS: The caveolae proteome in gentamicin treated cells shows that 40% of total proteins are uniquely associated with caveolae during the treatment, and 15% of the proteins normally associated with caveolae in untreated cell are suppressed. Bioinformatic analysis of the data shows a decreased expression of proteins involved in genetic information processing, and an increase in proteins involved in metabolism, vesicular transport and signal transduction in gentamicin treated cells. Several Rab GTPases proteins, ubiquitous transporters, uniquely segregate with caveolae and are significantly enriched in gentamicin treated cells. CONCLUSION: We report that gentamicin exposure modifies protein profile of caveolae from SL pericytes. We identified a pool of proteins which are uniquely segregating with caveolae during the treatment, mainly participating in metabolic and biosynthetic pathways, in transport pathways and in genetic information processing. Finally, we show for the first time proteins associated with caveolae SL pericytes linked to nonsyndromic hearing loss.

Rat airway smooth muscle cell during actin modulation: rheology and glassy dynamics.

Although changes of cytoskeleton (CSK) stiffness and friction can be induced by diverse interventions, all mechanical changes reported to date can be scaled onto master relationships that appear to be universal. To assess the limits of the applicability of those master relationships, we focused in the present study on actin and used a panel of actin-manipulating drugs that is much wider than any used previously. We focused on the cultured rat airway smooth muscle (ASM) cell as a model system. Cells were treated with agents that directly modulate the polymerization (jasplakinolide, cytochalasin D, and latrunculin A), branching (genistein), and cross linking (phallacidin and phalloidin oleate) of the actin lattice. Contractile (serotonin, 5-HT) and relaxing (dibutyryl adenosine 3',5'-cyclic monophosphate, DBcAMP) agonists and a myosin inhibitor (ML-7) were also tested for comparison, because these agents may change the structure of actin indirectly. Using optical magnetic twisting cytometry, we measured elastic and frictional moduli before and after treatment with each agent. Stiffness increased with frequency as a weak power law, and changes of friction paralleled those of stiffness until they approached a Newtonian viscous limit. Despite large differences in the mechanism of action among the interventions, all data collapsed onto master curves that depended on a single parameter. In the context of soft glassy systems, that parameter would correspond to an effective temperature of the cytoskeletal matrix and reflect the effects of molecular crowding and associated molecular trapping. These master relationships demonstrate that when the mechanical properties of the cell change, they are constrained to do so along a special trajectory. Because mechanical characteristics of the cell shadow underlying molecular events, these results imply special constraints on the protein-protein interactions that dominate CSK mechanical properties.

Time scale and other invariants of integrative mechanical behavior in living cells.

In dealing with systems as complex as the cytoskeleton, we need organizing principles or, short of that, an empirical framework into which these systems fit. We report here unexpected invariants of cytoskeletal behavior that comprise such an empirical framework. We measured elastic and frictional moduli of a variety of cell types over a wide range of time scales and using a variety of biological interventions. In all instances elastic stresses dominated at frequencies below 300 Hz, increased only weakly with frequency, and followed a power law; no characteristic time scale was evident. Frictional stresses paralleled the elastic behavior at frequencies below 10 Hz but approached a Newtonian viscous behavior at higher frequencies. Surprisingly, all data could be collapsed onto master curves, the existence of which implies that elastic and frictional stresses share a common underlying mechanism. Taken together, these findings define an unanticipated integrative framework for studying protein interactions within the complex microenvironment of the cell body, and appear to set limits on what can be predicted about integrated mechanical behavior of the matrix based solely on cytoskeletal constituents considered in isolation. Moreover, these observations are consistent with the hypothesis that the cytoskeleton of the living cell behaves as a soft glassy material, wherein cytoskeletal proteins modulate cell mechanical properties mainly by changing an effective temperature of the cytoskeletal matrix. If so, then the effective temperature becomes an easily quantified determinant of the ability of the cytoskeleton to deform, flow, and reorganize.