A membrane-based force generation mechanism in auditory sensory cells.

Auditory outer hair cells can elongate and shorten at acoustic frequencies in response to changes of plasma membrane potential. We show that this fast bidirectional contractile activity consists of an electromechanical transduction process that occurs at the lateral plasma membrane and can be activated and analyzed independently in small membrane patches inside a patch electrode. Bidirectional forces are generated by increases and decreases in membrane area in response to hyperpolarization and depolarization, respectively. We suggest that the force generation mechanism is driven by voltage-dependent conformational changes within a dense array of large transmembrane proteins associated with the site of electromechanical transduction.     

Motility voltage sensor of the outer hair cell resides within the lateral plasma membrane.

The outer hair cell (OHC) from the organ of Corti is believed to be responsible for the mammal's exquisite sense of hearing. A membrane-based motile response of this cell underlies the initial processing of acoustic energy. The voltage-dependent capacitance of the OHC, possibly reflecting charge movement of the motility voltage sensor, was measured in cells during intracellular dialysis of trypsin under whole cell voltage clamp. Within 10 min after dialysis, light and electron microscopic examination revealed that the subplasmalemmal structures, including the cytoskeletal framework and subsurface cisternae, were disrupted and/or detached from adjacent plasma membrane. Dialysis of heat-inactivated trypsin produced no changes in cell structure. Simultaneous measures of linear and nonlinear membrane capacitance revealed minimal changes, indicating that contributions by subsurface structures to the generation of the nonlinear capacitance are unlikely. This study strongly suggests that voltage-dependent charge movement in the OHC reflects properties of the force generator's voltage sensor and that the sensor/motor resides solely within the lateral plasma membrane.     

Molecular mechanisms of sound amplification in the mammalian cochlea

Mammalian hearing depends on the enhanced mechanical properties of the basilar membrane within the cochlear duct. The enhancement arises through the action of outer hair cells that act like force generators within the organ of Corti. Simple considerations show that underlying mechanism of somatic motility depends on local area changes within the lateral membrane of the cell. The molecular basis for this phenomenon is a dense array of particles that are inserted into the basolateral membrane and that are capable of sensing membrane potential field. We show here that outer hair cells selectively take up fructose, at rates high enough to suggest that a sugar transporter may be part of the motor complex. The relation of these findings to a recent candidate for the molecular motor is also discussed.     

Somatic stiffness of cochlear outer hair cells is voltage-dependent

The mammalian cochlea depends on an amplification process for its sensitivity and frequency-resolving capability. Outer hair cells are responsible for providing this amplification. It is usually assumed that the membrane-potential-driven somatic shape changes of these cells are the basis of the amplifying process. It is of interest to see whether mechanical reactance changes of the cells might accompany their changes in cell shape. We now show that the cylindrical outer hair cells change their axial stiffness as their membrane potential is altered. Cell stiffness was determined by optoelectronically measuring the amplitude of motion of a flexible vibrating fiber as it was loaded by the isolated cell. Voltage commands to the cell were delivered in a tight-seal whole-cell configuration. Cell stiffness was decreased by depolarization and increased by hyperpolarization.     

Receptor synapses without synaptic ribbons in the cochlea of the cat.

This report describes recepto-neural junctions thought to be chemical synapses without synaptic ribbons or vesicle aggregates. Such synapses occur in the cat between the outer hair cells of the cochlea and auditory nerve fibers. These synapses are formed by flat or indented junctions having symmetric membrane complexes associated with smooth endoplasmic reticulum and coated vesicles in the outer hair cell cytoplasm. Inner hair cells have receptoneural junctions with asymmetric membrane complexes; synaptic ribbons and vesicles gather at one type of junction, while the other type associates with endoplasmic reticulum. Gap junctions are not seen at cochlear synapses but do link supporting cells. The results suggest that inner and outer hair cell synapses have different properties and raise questions about the role of synaptic vesicles and endoplasmic reticulum in the storage and release of transmitters.     

The initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes

During oxygenic photosynthesis in cyanobacteria and chloroplasts of plants and eukaryotic algae, conversion of light energy to biologically useful chemical energy occurs in the specialized thylakoid membranes. Light-induced charge separation at the reaction centers of photosystems I and II, two multisubunit pigment-protein complexes in the thylakoid membranes, energetically drive sequential photosynthetic electron transfer reactions in this membrane system. In general, in the prokaryotic cyanobacterial cells, the thylakoid membrane is distinctly different from the plasma membrane. We have recently developed a two-dimensional separation procedure to purify thylakoid and plasma membranes from the genetically widely studied cyanobacterium Synechocystis sp. PCC 6803. Immunoblotting analysis demonstrated that the purified plasma membrane contained a number of protein components closely associated with the reaction centers of both photosystems. Moreover, these proteins were assembled in the plasma membrane as chlorophyll-containing multiprotein complexes, as evidenced from nondenaturing green gel and low-temperature fluorescence spectroscopy data. Furthermore, electron paramagnetic resonance spectroscopic analysis showed that in the partially assembled photosystem I core complex in the plasma membrane, the P700 reaction center was capable of undergoing light-induced charge separation. Based on these data, we propose that the plasma membrane, and not the thylakoid membrane, is the site for a number of the early steps of biogenesis of the photosynthetic reaction center complexes in these cyanobacterial cells.     

Uncoupling of transfer of the presequence and unfolding of the mature domain in precursor translocation across the mitochondrial outer membrane

Translocation of mitochondrial precursor proteins across the mitochondrial outer membrane is facilitated by the translocase of the outer membrane (TOM) complex. By using site-specific photocrosslinking, we have mapped interactions between TOM proteins and a mitochondrial precursor protein arrested at two distinct stages, stage A (accumulated at 0 degrees C) and stage B (accumulated at 30 degrees C), in the translocation across the outer membrane at high resolution not achieved previously. Although the stage A and stage B intermediates were assigned previously to the forms bound to the cis site and the trans site of the TOM complex, respectively, the results of crosslinking indicate that the presequence of the intermediates at both stage A and stage B is already on the trans side of the outer membrane. The mature domain is unfolded and bound to Tom40 at stage B whereas it remains folded at stage A. After dissociation from the TOM complex, translocation of the stage B intermediate, but not of the stage A intermediate, across the inner membrane was promoted by the intermembrane-space domain of Tom22. We propose a new model for protein translocation across the outer membrane, where translocation of the presequence and unfolding of the mature domain are not necessarily coupled.     

The origin of and subcellular mechanisms causing pancreatic bicarbonate secretion.

In recent years, there has been a rapid growth in knowledge about the subcellular mechanisms involved in pancreatic ductal secretion of bicarbonate. The mechanisms governing anion transport across the luminal membrane of duct cells have been well characterized. Evidence suggests that the cystic fibrosis transmembrane conductance regulator is a cyclic adenosine monophosphate-regulated Cl- conductance in the luminal membrane that plays a pivotal role in ductal bicarbonate secretion by recirculating the Cl- imported into duct cells through Cl(-)-HCO3- exchange. The mechanisms governing ion transfer across the basolateral plasma membrane of duct cells are less well defined. There is some evidence suggesting that secretin may cause exocytotic insertion of proton pumps into the basolateral plasma membrane. Once inserted into the plasma membrane, proton pumps could engage in primary active electrogenic H+ ion transport to interstitial tissue while secondary active HCO3- secretion occurs over the luminal membrane through Cl(-)-HCO3- exchangers coupled in parallel with the cystic fibrosis transmembrane conductance regulator. These and other subcellular phenomena related to ductal secretory function are reviewed.     

Resolving voltage-dependent structural changes of a membrane photoreceptor by surface-enhanced IR difference spectroscopy

Membrane proteins are molecular machines that transport ions, solutes, or information across the cell membrane. Electrophysiological techniques have unraveled many functional aspects of ion channels but suffer from the lack of structural sensitivity. Here, we present spectroelectrochemical data on vibrational changes of membrane proteins derived from a single monolayer. For the seven-helical transmembrane protein sensory rhodopsin II, structural changes of the protein backbone and the retinal cofactor as well as single ion transfer events are resolved by surface-enhanced IR difference absorption spectroscopy (SEIDAS). Angular changes of bonds versus the membrane normal have been determined because SEIDAS monitors only those vibrations whose dipole moment are oriented perpendicular to the solid surface. The application of negative membrane potentials (ΔV = −0.3 V) leads to the selective halt of the light-induced proton transfer at the stage of D75, the counter ion of the retinal Schiff base. It is inferred that the voltage raises the energy barrier of this particular proton-transfer reaction, rendering the energy deposited in the retinal by light excitation insufficient for charge transfer to occur. The other structural rearrangements that accompany light-induced activity of the membrane protein, are essentially unaffected by the transmembrane electric field. Our results demonstrate that SEIDAS is a generic approach to study processes that depend on the membrane potential, like those in voltage-gated ion channels and transporters, to elucidate the mechanism of ion transfer with unprecedented spatial sensitivity and temporal resolution.     

Role of deformation-induced lipid trafficking in the prevention of plasma membrane stress failure

Cells experience plasma membrane stress failure when the matrix to which they adhere undergoes large deformations. In the lung, such a mechanism might explain mechanical ventilation-associated cell injury. We have previously shown that in alveolar epithelial cells, deformation induces lipid trafficking to the plasma membrane, thereby accommodating the required increase in the cell surface area. We now show that cell wounding is strain amplitude and rate dependent and that under conditions of impaired exocytosis strain-induced cell wounding is significantly increased. In addition, the susceptibility of cells to mechanical injury was not correlated with changes in cell stiffness. Using a dual-labeling technique, we differentiated between cell populations that were reversibly and irreversibly injured and showed that interventions that impair deformation-induced lipid trafficking also reduce the likelihood of plasma membrane resealing. Our findings suggest that cell plasticity and remodeling responses such as deformation-induced lipid trafficking are more important for cytoprotection from strain injury than are the innate mechanical properties of the cell. We also conclude that in deformation experiments, tests of cell membrane integrity cannot be interpreted as tests of cell viability because an intact plasma membrane after deformation does not mean that no injury had occurred.     

FAM65B is a membrane-associated protein of hair cell stereocilia required for hearing

In a large consanguineous Turkish kindred with recessive nonsyndromic, prelingual, profound hearing loss, we identified in the gene FAM65B (MIM611410) a splice site mutation (c.102-1G>A) that perfectly cosegregates with the phenotype in the family. The mutation leads to exon skipping and deletion of 52-amino acid residues of a PX membrane localization domain. FAM65B is known to be involved in myotube formation and in regulation of cell adhesion, polarization, and migration. We show that wild-type Fam65b is expressed during embryonic and postnatal development stages in murine cochlea, and that the protein localizes to the plasma membranes of the stereocilia of inner and outer hair cells of the inner ear. The wild-type protein targets the plasma membrane, whereas the mutant protein accumulates in cytoplasmic inclusion bodies and does not reach the membrane. In zebrafish, knockdown of fam65b leads to significant reduction of numbers of saccular hair cells and neuromasts and to hearing loss. We conclude that FAM65B is a plasma membrane-associated protein of hair cell stereocilia that is essential for hearing.Hearing loss is the most common sensory problem, affecting approximately 1 in 500 newborns. Most cases are the consequence of mutations in single genes with specific functions in the inner ear (1) (http://hereditaryhearingloss.org). Hearing depends on the ability of the inner ear to convert acoustic waves into electrical signals. This process originates in the stereocilia, actin-rich structures that project from the apical pole of cochlear hair cells and are interconnected in the shape of a staircase to form the hair bundle. Most of the ∼50 hair-bundle proteins identified so far are the products of genes that when mutated lead to hearing loss (2). Thus, the genetic approach has played a major role in elucidating the molecular components of normal hearing.Here we present Family With Sequence Similarity 65, Member B (FAM65B, MIM611410) as a previously unrecognized, plasma membrane-associated protein of hair cell stereocilia. The critical role of FAM65B in human hearing was revealed by genetic analysis of a large family with hereditary deafness. In the zebrafish, knocking down the ortholog of FAM65B led to sensorineural hearing loss.     

The 3D structure of a periplasm-spanning platform required for assembly of group 1 capsular polysaccharides in Escherichia coli

Capsular polysaccharides (CPSs) are essential virulence determinants of many pathogenic bacteria. Escherichia coli group 1 CPSs provide paradigms for widespread surface polysaccharide assembly systems in Gram-negative bacteria. In these systems, complex carbohydrate polymers must be exported across the periplasm and outer membrane to the cell surface. Group 1 CPS export requires oligomers of the outer membrane protein, Wza, for translocation across the outer membrane. Assembly also depends on Wzc, an inner membrane tyrosine autokinase known to regulate export and synthesis of group 1 CPS. Here, we provide a structural view of a complex comprising Wzc and Wza that spans the periplasm, connecting the inner and outer membranes. Examination of transmembrane sections of the complex suggests that the periplasm is compressed at the site of complex formation. An important feature of CPS production is the coupling of steps involved in biosynthesis and export. We propose that the Wza-Wzc complex provides the structural and regulatory core of a larger macromolecular machine. We suggest a mechanism by which CPS may move from the periplasm through the outer membrane.     

Estradiol-induced changes in progesterone secretion by rabbit corpora lutea are associated with quantitative ultrastructural changes in luteal cells

In this study, we examined changes in luteal cell structure that accompany estradiol-altered progesterone production by the rabbit corpus luteum. To stimulate progesterone production, polydimethylsiloxane (Silastic) capsules containing 17 beta-estradiol were inserted sc into superovulated New Zealand White rabbits. Luteal progesterone production, assessed by measurement of progesterone in peripheral serum, was high after the estradiol-filled capsules were inserted, declined within 24 h after the capsules were removed, and increased within 32 h after reinsertion of the capsules. Stereological analyses at the light microscopic level revealed that the number of luteal cells and the volume of an average luteal cell did not differ significantly between estradiol-stimulated and estradiol-deprived rabbits over the time period employed. In contrast, stereological analyses at the electron microscopic level demonstrated that the surface areas of smooth endoplasmic reticulum and inner mitochondrial membrane declined with estradiol deprivation, but were restored by reimposition of estradiol stimulation. These changes in the surface area per cell of smooth endoplasmic reticulum and inner mitochondrial membrane were strongly correlated (r = 0.94 and r = 0.88, respectively) with changes in progesterone concentrations in peripheral serum. Changes in the surface area of lipid droplets per luteal cell also occurred, but were inversely correlated (r = -0.87) with progesterone levels. No significant changes were seen in the surface areas per cell of outer mitochondrial membrane or rough endoplasmic reticulum. These results demonstrate that estradiol stimulation and deprivation cause reversible quantitative changes in the rabbit luteal cell organelles known to be directly involved in progesterone biosynthesis. This leads to the conclusion that the steroidogenic activity of the luteal cell is tightly coupled to its subcellular structure.     

Tension in secretory granule membranes causes extensive membrane transfer through the exocytotic fusion pore.

For fusion to occur the repulsive forces between two interacting phospholipid bilayers must be reduced. In model systems, this can be achieved by increasing the surface tension of at least one of the membranes. However, there has so far been no evidence that the secretory granule membrane is under tension. We have been studying exocytosis by using the patch-clamp technique to measure the surface area of the plasma membrane of degranulating mast cells. When a secretory granule fuses with the plasma membrane there is a step increase in the cell surface area. Some fusion events are reversible, in which case we have found that the backstep is larger than the initial step, indicating that there is a net decrease in the area of the plasma membrane. The decrease has the following properties: (i) the magnitude is strongly dependent on the lifetime of the fusion event and can be extensive, representing as much as 40% of the initial granule surface area; (ii) the rate of decrease is independent of granule size; and (iii) the decrease is not dependent on swelling of the secretory granule matrix. We conclude that the granule membrane is under tension and that this tension causes a net transfer of membrane from the plasma membrane to the secretory granule, while they are connected by the fusion pore. The high membrane tension in the secretory granule may be the critical stress necessary for bringing about exocytotic fusion.     

Spectrin,red cell shape and deformability

Summary In a companion paper, the shapes of spectrin deficient mouse erythrocytes were described; in contrast to previous assumptions, spherules with tethered microvesicles rather than true spherocytes were found. Thence, spectrin deficient mouse erythrocytes are endowed with an excess of surface area for the given volume but the membrane is assuming a highly positive curvature. Observations during and after the action of enzymes cleaving the red cell surface charge (Neuraminidase, Trypsin, Chymotrypsin) showed that the previously positive membrane curvature, as well as the tendency of the membrane to flow into fingerlike protrusions was completely abolished. The erythrocytes of the spectrin deficient, desialylated mouse erythrocytes assumed a variety of shapes, often discocytic or even stomatocytic, i.e. their membrane presented with negative curvature. However, while these desialylated membranes could be easily deformed (elongated) by shear flow they did not recoil elastically into any definitive configuration after removal of the deforming forces. It is concluded from these observations that spectrin (acting on the inner interface between membrane and cytoplasm) and sialic acid residues (acting on the outer interface between membrane and plasma) exert antagonizing effects on membrane curvature and membrane bending elasticity. Sialic acid residues, strongly charged and situated on the outer side of the cell, produce positive membrane curvature; this observation can most readily be explained by assuming that this mechanical effect is caused by repulsive coulombic forces expanding the outer half of the bilayer. To explain the effect of the spectrin-complex in counteracting positive or in producing negative membrane curvature, a similar expansive coulombic force acting between the highly charged residues has been postulated. Thence, a model for explaining the overall elastic behaviour of the normal mammalian red cell is developed which is based on the assumption of elastic interactions of proteinacous membrane components coupled to the lipid bilayer of the membrane.Supported by Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 109, Project C 7. Dedicated to the memory of the late Professor Marcel Hanns, who not only made possible the measurements of zeta-potential in the present experiments but who, in many discussions of the scientific problems envolved, was very instrumental in shaping the concepts presented in this communication.     

Changes in intra- or extracellular pH do not mediate P-glycoprotein-dependent multidrug resistance.

P-glycoprotein (Pgp)-mediated multidrug resistance (MDR) is thought to result from active extrusion of lipid-soluble, titratable chemotherapeutic agents. Given the lack of demonstration of coupling between ATP hydrolysis and drug transport, the resistance to chemically unrelated compounds, and findings of elevated intracellular pH (pHi), it has been proposed that reduced intracellular accumulation of drugs in MDR is due to changes in the pH difference across the plasma membrane. Elevation of pHi or decrease in local extracellular pH (pHo) could reduce the intracellular accumulation of the protonated chemotherapeutic drugs and account for Pgp-mediated MDR. Alternatively, changes in pHi or pHo could increase drug efflux by other mechanisms, such as coupled transport involving H+ or OH-, or allosteric effects on Pgp or other proteins. Both mechanisms could operate independently of the charge of the substrate. The possibility of a role of pHi in drug efflux is important to test because of the clinical significance of the phenomenon of MDR of tumors. We tested this hypothesis and found that MDR can occur in cells with low, normal, or high pHi. Further, resistant cells exhibited reduced steady-state drug accumulation and increased efflux without changes in local pHo. Finally, acute changes in pHi had no appreciable effect on Pgp-mediated drug efflux. We conclude that Pgp-mediated MDR is not a consequence of changes in pHi or pHo.     

Endosomal recycling controls plasma membrane area during mitosis

The shape and total surface of a cell and its daughters change during mitosis. Many cells round up during prophase and metaphase and reacquire their extended and flattened shape during cytokinesis. How does the total area of plasma membrane change to accommodate these morphological changes and by what mechanism is control of total membrane area achieved? Using single-cell imaging methods, we have found that the amount of plasma membrane in attached cells in culture decreases at the beginning of mitosis and recovers rapidly by the end. Clathrin-based endocytosis is normal throughout all phases of cell division, whereas recycling of internalized membranes back to the cell surface slows considerably during the rounding up period and resumes at the time at which recovery of cell membrane begins. Interference with either one of these processes by genetic or chemical means impairs cell division. The total cell-membrane area recovers even in the absence of a functional Golgi apparatus, which would be needed for export of newly synthesized membrane lipids and proteins. We propose a mechanism by which modulation of endosomal recycling controls cell area and surface expression of membrane-bound proteins during cell division.     

Two distinct mechanisms drive protein translocation across the mitochondrial outer membrane in the late step of the cytochrome b2 import pathway

The import of cytochrome b(2) into mitochondria consists of two steps. The translocation of the first part of the presequence across the inner membrane is coupled with the translocation of the tightly folded heme-binding domain across the outer membrane and requires a membrane potential DeltaPsi and the functions of mitochondrial Hsp70 (mHsp70) in the matrix. Once the heme-binding domain has passed the outer membrane, the translocation of the rest of the polypeptide chain across the outer membrane becomes independent of DeltaPsi and mHsp70. Here we analyzed the late DeltaPsi- and mHsp70-independent step in the transport of cytochrome b(2) fusion proteins into the intermembrane space (IMS). The import of the cytochrome b(2) fusion proteins containing two protein domains linked by a spacer segment into mitochondria was arrested at a stage at which one domain folded on each side of the outer membrane, along the pathway that is consistent with the stop-transfer model. The mature-size form of the translocation intermediate could move across the outer membrane in both directions, and the stabilization of the protein domain in the IMS promoted the forward translocation. On the other hand, the intermediate-size form of the translocation intermediate, which retains the anchorage to the inner membrane, was transported into the IMS independently of the stability of the protein domain in the IMS. These results suggest that two distinct mechanisms, the Brownian ratchet and the anchor diffusion mechanisms, can operate for the transmembrane movement of the mature-size form and the intermediate-size form, respectively, of cytochrome b(2) species.     

Striated organelle, a cytoskeletal structure positioned to modulate hair-cell transduction

The striated organelle (SO), a cytoskeletal structure located in the apical region of cochlear and vestibular hair cells, consists of alternating, cross-linked, thick and thin filamentous bundles. In the vestibular periphery, the SO is well developed in both type I and type II hair cells. We studied the 3D structure of the SO with intermediate-voltage electron microscopy and electron microscope tomography. We also used antibodies to α-2 spectrin, one protein component, to trace development of the SO in vestibular hair cells over the first postnatal week. In type I cells, the SO forms an inverted open-ended cone attached to the cell membrane along both its upper and lower circumferences and separated from the cuticular plate by a dense cluster of exceptionally large mitochondria. In addition to contacts with the membrane and adjacent mitochondria, the SO is connected both directly and indirectly, via microtubules, to some stereociliary rootlets. The overall architecture of the apical region in type I hair cells--a striated structure restricting a cluster of large mitochondria between its filaments, the cuticular plate, and plasma membrane--suggests that the SO might serve two functions: to maintain hair-cell shape and to alter transduction by changing the geometry and mechanical properties of hair bundles.