Stereocilia-staircase spacing is influenced by myosin III motors and their cargos espin-1 and espin-like

Hair cells tightly control the dimensions of their stereocilia, which are actin-rich protrusions with graded heights that mediate mechanotransduction in the inner ear. Two members of the myosin-III family, MYO3A and MYO3B, are thought to regulate stereocilia length by transporting cargos that control actin polymerization at stereocilia tips. We show that eliminating espin-1 (ESPN-1), an isoform of ESPN and a myosin-III cargo, dramatically alters the slope of the stereocilia staircase in a subset of hair cells. Furthermore, we show that espin-like (ESPNL), primarily present in developing stereocilia, is also a myosin-III cargo and is essential for normal hearing. ESPN-1 and ESPNL each bind MYO3A and MYO3B, but differentially influence how the two motors function. Consequently, functional properties of different motor-cargo combinations differentially affect molecular transport and the length of actin protrusions. This mechanism is used by hair cells to establish the required range of stereocilia lengths within a single cell

Stereocilia-staircase spacing is influenced by myosin III motors and their cargos espin-1 and espin-like

Hair cells tightly control the dimensions of their stereocilia, which are actin-rich protrusions with graded heights that mediate mechanotransduction in the inner ear. Two members of the myosin-III family, MYO3A and MYO3B, are thought to regulate stereocilia length by transporting cargos that control actin polymerization at stereocilia tips. We show that eliminating espin-1 (ESPN-1), an isoform of ESPN and a myosin-III cargo, dramatically alters the slope of the stereocilia staircase in a subset of hair cells. Furthermore, we show that espin-like (ESPNL), primarily present in developing stereocilia, is also a myosin-III cargo and is essential for normal hearing. ESPN-1 and ESPNL each bind MYO3A and MYO3B, but differentially influence how the two motors function. Consequently, functional properties of different motor-cargo combinations differentially affect molecular transport and the length of actin protrusions. This mechanism is used by hair cells to establish the required range of stereocilia lengths within a single cell

Stereocilia-staircase spacing is influenced by myosin III motors and their cargos espin-1 and espin-like

Hair cells tightly control the dimensions of their stereocilia, which are actin-rich protrusions with graded heights that mediate mechanotransduction in the inner ear. Two members of the myosin-III family, MYO3A and MYO3B, are thought to regulate stereocilia length by transporting cargos that control actin polymerization at stereocilia tips. We show that eliminating espin-1 (ESPN-1), an isoform of ESPN and a myosin-III cargo, dramatically alters the slope of the stereocilia staircase in a subset of hair cells. Furthermore, we show that espin-like (ESPNL), primarily present in developing stereocilia, is also a myosin-III cargo and is essential for normal hearing. ESPN-1 and ESPNL each bind MYO3A and MYO3B, but differentially influence how the two motors function. Consequently, functional properties of different motor-cargo combinations differentially affect molecular transport and the length of actin protrusions. This mechanism is used by hair cells to establish the required range of stereocilia lengths within a single cell

Freeze-Fracture Replica Immunolabelling Reveals Urothelial Plaques in Cultured Urothelial Cells

The primary function of the urothelium is to provide the tightest and most impermeable barrier in the body, i.e. the blood-urine barrier. Urothelial plaques are formed and inserted into the apical plasma membrane during advanced stages of urothelial cell differentiation. Currently, it is supposed that differentiation with the final formation of urothelial plaques is hindered in cultured urothelial cells. With the aid of the high-resolution imaging technique of freeze-fracture replica immunolabelling, we here provide evidence that urothelial cells in vitro form uroplakin-positive urothelial plaques, localized in fusiform-shaped vesicles and apical plasma membranes. With the establishment of such an in vitro model of urothelial cells with fully developed urothelial plaques and functional properties equivalent to normal bladder urothelium, new perspectives have emerged which challenge prevailing concepts of apical plasma membrane biogenesis and blood-urine barrier development. This may hopefully provide a timely impulse for many ongoing studies and open up new questions for future research

Protein Kinase C Activation Has Distinct Effects on the Localization, Phosphorylation and Detergent Solubility of the Claudin Protein Family in Tight and Leaky Epithelial Cells

We have previously shown that protein kinase C (PKC) activation has distinct effects on the structure and barrier properties of cultured epithelial cells (HT29 and MDCK I). Since the claudin family of tight junction (TJ)-associated proteins is considered to be crucial for the function of mature TJ, we assessed their expression patterns and cellular destination, detergent solubility and phosphorylation upon PKC stimulation for 2 or 18 h with phorbol myristate acetate (PMA). In HT29 cells, claudins 1, 3, 4 and 5 and possibly claudin 2 were redistributed to apical cell–cell contacts after PKC activation and the amounts of claudins 1, 3 and 5, but not of claudin 2, were increased in cell lysates. By contrast, in MDCK I cells, PMA treatment resulted in redistribution of claudins 1, 3, 4 and 5 from the TJ and in reorganization of the proteins into more insoluble complexes. Claudins 1 and 4 were phosphorylated in both MDCK I and HT29 cells, but PKC-induced changes in claudin phosphorylation state were detected only in MDCK I cells. A major difference between HT29 and MDCK I cells, which have low and high basal transepithelial electrical resistance, respectively, was the absence of claudin 2 in the latter. Our findings show that PKC activation targets in characteristic ways the expression patterns, destination, detergent solubility and phosphorylation state of claudins in epithelial cells with different capacities to form an epithelial barrier

Topology of molecular machines of the endoplasmic reticulum: a compilation of proteomics and cytological data

The endoplasmic reticulum (ER) is a key organelle of the secretion pathway involved in the synthesis of both proteins and lipids destined for multiple sites within and without the cell. The ER functions to both co- and post-translationally modify newly synthesized proteins and lipids and sort them for housekeeping within the ER and for transport to their sites of function away from the ER. In addition, the ER is involved in the metabolism and degradation of specific xenobiotics and endogenous biosynthetic products. A variety of proteomics studies have been reported on different subcompartments of the ER providing an ER protein dictionary with new data being made available on many protein complexes of relevance to the biology of the ER including the ribosome, the translocon, coatomer proteins, cytoskeletal proteins, folding proteins, the antigen-processing machinery, signaling proteins and proteins involved in membrane traffic. This review examines proteomics and cytological data in support of the presence of specific molecular machines at specific sites or subcompartments of the ER

Power efficiency of outer hair cell somatic electromotility

© 2009 Rabbitt et al. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in PLoS Computational Biology 5 (2009): e1000444, doi:10.1371/journal.pcbi.1000444.Cochlear outer hair cells (OHCs) are fast biological motors that serve to enhance the vibration of the organ of Corti and increase the sensitivity of the inner ear to sound. Exactly how OHCs produce useful mechanical power at auditory frequencies, given their intrinsic biophysical properties, has been a subject of considerable debate. To address this we formulated a mathematical model of the OHC based on first principles and analyzed the power conversion efficiency in the frequency domain. The model includes a mixture-composite constitutive model of the active lateral wall and spatially distributed electro-mechanical fields. The analysis predicts that: 1) the peak power efficiency is likely to be tuned to a specific frequency, dependent upon OHC length, and this tuning may contribute to the place principle and frequency selectivity in the cochlea; 2) the OHC power output can be detuned and attenuated by increasing the basal conductance of the cell, a parameter likely controlled by the brain via the efferent system; and 3) power output efficiency is limited by mechanical properties of the load, thus suggesting that impedance of the organ of Corti may be matched regionally to the OHC. The high power efficiency, tuning, and efferent control of outer hair cells are the direct result of biophysical properties of the cells, thus providing the physical basis for the remarkable sensitivity and selectivity of hearing.This work was supported by NIDCD R01 DC04928 (Rabbitt), NIDCD R01 DC00384 (Brownell) and NASA Ames GSRA56000135 (Breneman)

Oligodendrocyte Development in the Absence of Their Target Axons In Vivo

Oligodendrocytes form myelin around axons of the central nervous system, enabling saltatory conduction. Recent work has established that axons can regulate certain aspects of oligodendrocyte development and myelination, yet remarkably oligodendrocytes in culture retain the ability to differentiate in the absence of axons and elaborate myelin sheaths around synthetic axon-like substrates. It remains unclear the extent to which the life-course of oligodendrocytes requires the presence of, or signals derived from axons in vivo. In particular, it is unclear whether the specific axons fated for myelination regulate the oligodendrocyte population in a living organism, and if so, which precise steps of oligodendrocyte-cell lineage progression are regulated by target axons. Here, we use live-imaging of zebrafish larvae carrying transgenic reporters that label oligodendrocyte-lineage cells to investigate which aspects of oligodendrocyte development, from specification to differentiation, are affected when we manipulate the target axonal environment. To drastically reduce the number of axons targeted for myelination, we use a previously identified kinesin-binding protein (kbp) mutant, in which the first myelinated axons in the spinal cord, reticulospinal axons, do not fully grow in length, creating a region in the posterior spinal cord where most initial targets for myelination are absent. We find that a 73% reduction of reticulospinal axon surface in the posterior spinal cord of kbp mutants results in a 27% reduction in the number of oligodendrocytes. By time-lapse analysis of transgenic OPC reporters, we find that the reduction in oligodendrocyte number is explained by a reduction in OPC proliferation and survival. Interestingly, OPC specification and migration are unaltered in the near absence of normal axonal targets. Finally, we find that timely differentiation of OPCs into oligodendrocytes does not depend at all on the presence of target axons. Together, our data illustrate the power of zebrafish for studying the entire life-course of the oligodendrocyte lineage in vivo in an altered axonal environment

Tight junctions: from simple barriers to multifunctional molecular gates

Epithelia and endothelia separate different tissue compartments and protect multicellular organisms from the outside world. This requires the formation of tight junctions, selective gates that control paracellular diffusion of ions and solutes. Tight junctions also form the border between the apical and basolateral plasma-membrane domains and are linked to the machinery that controls apicobasal polarization. Additionally, signalling networks that guide diverse cell behaviours and functions are connected to tight junctions, transmitting information to and from the cytoskeleton, nucleus and different cell adhesion complexes. Recent advances have broadened our understanding of the molecular architecture and cellular functions of tight junctions