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B Kachar

Publications and source records attributed to B Kachar.

At least 19 recordsLinked to original sources
PubMed1997

Compartmentalized vesicular traffic around the hair cell cuticular plate.

Through thin-section and freeze-fracture electron microscopy, we identify structural correlates of an intense vesicular traffic in a narrow band of cytoplasm around the cuticular plate of the bullfrog vestibular hair cells. Myriads of coated and uncoated vesicles associated with longitudinally oriented microtubules populate the narrow cytoplasmic region between the cuticular plate and the actin network of the apical junctional belt. If microtubules in the sensory hair cells, like those in axons, are pathways for organelle transport, then the characteristic distribution of microtubules around the cuticular plate represents transport pathways across the apical region of the hair cells. This compartmentalized membrane traffic system appears to support an intense vesicular release and uptake along a band of apical plasma membrane near the cell border. Functions of this transport system may include membrane recycling as well as exocytotic and endocytotic exchange between the hair cell cytoplasm and the endolymphatic compartment.

Acoustic Maculae↗
PubMed1996

Freeze-substitution as a preparative technique for immunoelectronmicroscopy: evaluation by atomic force microscopy.

Cryofixation followed by freeze substitution in osmium tetroxide was evaluated as a method for preparing biological specimens for immunoelectronmicroscopy. Samples were rapidly frozen by impact onto a sapphire block cooled with liquid nitrogen, substituted at -80 degrees C in acetone containing osmium tetroxide, and embedded in epoxy resin. With this protocol, excellent ultrastructure can be combined with localization of antigens that otherwise would be inactivated by the osmium, but labeling may need to be enhanced by chemically etching the sections prior to staining. The effects of etching on various structures in the sections were investigated by examining the sections with atomic force microscopy, an approach that yields three-dimensional views of the surface of the section. A considerable part of the section was removed or collapsed by the etching, and these effects occurred differentially in several components of the tissue and with different etching protocols. Nevertheless, the results suggest that the partial removal of the plastic by etching of freeze-substituted tissue can be explored as a method for exposing fine biological structures for observation with atomic force microscopy.

Animals↗
PubMed1996

Identification of isoforms of G proteins and PKC that colocalize with tight junctions.

Recent evidence suggests that the formation and permeability of tight junctions are actively regulated by second-messenger-generating systems involving G proteins and protein kinase C (PKC). A possible specific target for these regulatory proteins is the tight junction protein ZO-1. An extensive immunocytochemical study was performed in cultured epithelial monolayers of MDCK and Caco-2 cells to identify which isoforms of G proteins and PKC are present at or near zonula occludens complex. Antibodies against alpha-subunits of each one of the four major subfamilies were used for the localization of the G proteins. For the PKC localization, antibodies against eight different isoforms were used. In confluent monolayers, G alpha 12 and PKC zeta, were the only isoforms of these proteins at the cell borders. In subconfluent monolayers, G alpha 12 and PKC zeta were found at the plasma membrane only along the areas of lateral cell-cell contact. These isoforms formed a pattern of distribution very similar to the ZO-1 protein. The present findings indicate that G alpha 12 and PKC zeta may be part of the zonula occludens complex and may locally regulate formation and permeability of tight junctions.

Animals↗
PubMed1996

Tight junction dynamics in the frog urinary bladder.

In a previous study in frog skin (Castro et al., J. Memb. Biol. 134:15-29, 1993), it was shown that TJs experimentally disrupted by a selective deposition of BaSO4 could be resealed upon addition of Ca2+ to the apical solution; in the absence of apical Ca2+, the normal Ca2+ activity of the Na2SO4-Ringer's bathing the basolateral side was not able to induce TJ resealing. We now show that apical Ca2+ also activates the TJ sealing mechanism in frog urinary bladders. Three known procedures were utilized to increase TJ permeability, all in the absence of apical Ca2+: (i) exposure to high positive transepithelial clamping potentials; (ii) exposure of the apical surface to hypertonic solutions; and (iii) selective deposition of BaSO4 in the TJs. The resealing of the TJs was promoted by raising the concentration of Ca2+ in the apical solution. This effect of Ca2+ is not impaired by the presence of Ca2+ channel blockers (nifedipine, verapamil, Mn2+ or Cd2+) in the apical solution, indicating that junction resealing does not depend on Ca2+ entering the cells through the apical membrane. TJ resealing that occurs in response to raised apical Ca2+ most likely results from a direct effect of Ca2+, entering the disrupted TJs from the apical solution and reaching the zonula adhaerens Ca2+ receptors (E-cadherins). Protein kinase C (PKC) must play a significant role in the control of TJ assembly in this tight epithelia since the PKC inhibitor (H7) and the activator (diC8) markedly affect TJ recovery after disruption by apical hypertonicity. H7 treated tissues show marked recuperation of conductance even in the absence of apical Ca2+. In contrast, diC8 prevents tissue recuperation which normally occurs after addition of Ca2+ to the apical solution.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1995

A soluble motor from the alga Nitella supports fast movement of actin filaments in vitro.

In the streaming cytoplasm of the Characean algae cell, the movement of organelles along actin bundles occurs at a striking rate of up to 60 microns s-1. To further characterize the molecular mechanisms responsible for this phenomenon, we have reconstituted the movement of actin filaments in vitro using defined biochemical components. We report that only a soluble cytoplasmic fraction devoid of organelles and filamentous material supports the movement of fluorescent-labeled actin filaments on glass at a rate of up to 60 microns s-1. This fraction also contains the K(+)-EDTA ATPase and the actin-activated Mg2+ ATPase activities characteristic of myosin proteins. Therefore, on the basis of these observations, we conclude that Nitella cells have a soluble pool of non-filamentous myosin molecules with the mechanochemical properties expected for a motor responsible for cytoplasmic streaming in vivo. The preparation and conditions described here should be useful for the purification of this translocator.

Actins↗
PubMed1995

Cloning of an organ of Corti anion exchanger 2 isoform with a truncated C-terminal domain.

We have isolated a cDNA clone from a guinea pig organ of Corti library encoding a new isoform of the Anion Exchanger 2 (AE2) protein. This cDNA clone shows an 83 bp deletion in the region that encodes the membrane domain of AE2. Analysis of the overlapping regions of genomic and cDNA clones indicates that the missing portion does not correspond exactly to a constitutive exon. The alternate splicing process that generates this transcript involves internal donor and acceptor sites which introduces a shift in the open reading frame. The resulting polypeptide has a conserved cytoplasmic N-terminal domain but the membrane C-terminal domain has only two of the fourteen membrane spanning regions. An affinity-purified antipeptide antibody to the novel C-terminus detects an 89 kDa polypeptide which agrees with the molecular mass predicted from the cDNA.

Amino Acid Sequence↗
PubMed1995

Linear gap and tight junctional assemblies between capillary endothelial cells in the eel rete mirabile.

BACKGROUND: Interendothelial tight junctions and gap junctions have been described in large blood vessels and in cultures of endothelium derived from large blood vessels. Transfer of microinjected small-molecular weight tracers between adjacent endothelial cells also has been demonstrated indicating the presence of gap junctional interendothelial communication. Similar transfer of tracers is evident between microvessel endothelial cells in culture and in microvessels in situ. However, gap junctions have not been detectable by electron microscopy of intact capillary systems. This may be due to limited sampling available in diffuse capillary systems and a small area of overlap between adjacent endothelial membranes. METHODS: Thin slices of the parallel, tightly packed capillary bed of the eel rete mirabile were cryofixed and prepared for conventional TEM by freeze-substitution. Other samples were freeze-fractured and replicated for examination of endothelial junctional components. RESULTS: A novel tight-gap junctional complex between rete capillary endothelial cells is described. In freeze-fracture replicas of the membrane P face, rows of gap junction subunits are flanked on either side by linear depressions representing grooves previously occupied by tight junctional strands that partition to the E face. In thin sections, the junctions appear in profile as short lengths of closely apposed membranes characteristic of gap junctions. CONCLUSIONS: The tight junctional components imply a barrier to paracellular transport across the capillary wall between the endothelial cells. The gap junctional component may provide a mechanism for communication between endothelial cells along the length of the vessel wall.

Air Sacs↗
PubMed1995

Analysis of the interdependent localization of vimentin and microtubules in neoplastic myoepithelial cells.

Salivary gland neoplastic myoepithelial cells in culture form very thin cytoplasmic processes in which the vimentin network is well dispersed. These vimentin filaments can be individually visualized by immunofluorescence. In this study, we have analyzed the role of microtubules in the distension and organization of the vimentin filament network found in these cells. We find that vimentin filaments colocalize along microtubules; however, a significant number of filaments can also be found in microtubule-free domains. Additionally, vimentin filaments are absent from large domains of microtubule-rich domains. Treatment of neoplastic myepithelial cells with the microtubule inhibitor nocodazole did not cause any retraction of the distended vimentin network. This observation suggests that the structural integrity of microtubules is not important for the stability of the vimentin network. Combining procedures for transient disruption of vimentin filaments and microtubules we observed that, in the absence of microtubules, the vimentin network could reassemble in the perinuclear region but was unable to extend toward the cell periphery. The dispersion of vimentin filaments to the peripheral regions of the cytoplasm could only be observed upon microtubule reassembly. This indicates that microtubules are not required for the stability of the vimentin network, but the dispersion of vimentin filaments to the peripheral cytoplasm depends on active interactions with microtubules.

Adenoma, Pleomorphic↗
PubMed1994

Structural basis for mechanical transduction in the frog vestibular sensory apparatus: II. The role of microtubules in the organization of the cuticular plate.

The actin matrix of the cuticular plate, which supports the sensory stereocilia bundle, is coupled to the axial cytoskeleton of the hair cell through a well defined microtubule columnar framework. A collection of axial microtubules in a columnar organization penetrate deep into the dense actin matrix of the cuticular plate. Each microtubule displays at the end a 300-500 nm long fuzzy cap that enmeshes with the actin matrix of the cuticular plate. The microtubule associated proteins MAP-1A and MAP-1B were localized by confocal immunofluorescence to the point of microtubule insertion in the cuticular plate. These proteins are likely components of the microtubule capping structure and may mediate the interaction of the microtubules with the actin matrix. The structural interaction of the microtubules with the cuticular plate provides important mechanical coupling of the transduction apparatus to the axial cytoskeleton of the hair cell.

Actins↗
PubMed1994

Modulation of tight junction morphology and permeability by an epithelial factor.

We report evidence of a factor secreted at the apical side of epithelial monolayers which modulates tight junction structure and permeability. This activity was detected within 4-7 days of conditioning of the apical medium by MDCK, A6 or Caco-2 epithelial cell lines cultured on permeable membranes in bipartite chambers. Apical conditioned medium (ACM), applied to the basolateral surface of a confluent monolayer, increased the transepithelial electrical resistance (TER), progressively reaching values 12-22% higher than the baseline within 5-10 min. After 40-60 min, the TER returned slowly to the basal value. This phenomenon was not observed either when using preheated ACM or the ACM filtrate obtained through a 30,000 MW cutoff membrane. The ACM maintained its activity even when applied to cell lines from different organs and species, as demonstrated when ACM from MDCK monolayers promoted an increase of 22% in the TER of Caco-2 cells. The increase of TER induced by the ACM treatment is accompanied by a change in the distribution of the number of tight junction strands, from an initial pattern, dominated mostly by junctions with one or two strands, to a new pattern after treatment dominated by junctions with two or three strands. Our results suggest the existence of a mechanism in epithelial cells that could signal leakage of apically secreted components to the basolateral side, thereby modulating the junction structure and permeability.

Animals↗
PubMed1993

Inhibition of outer hair cell electromotility by sulfhydryl specific reagents.

Mammalian outer hair cells can change length at acoustic frequencies when they are electrically stimulated. It was postulated that these length changes depend on electromechanical transduction based on voltage dependent conformational changes in a membrane motor protein. In this report, we describe the effect of various sulfhydryl (SH)-specific reagents on the OHC electromotility. p-Chloromercuriphenylsulfonate (pCMPS), in addition to other mercurials that can react with well-protected SH-groups in proteins, inhibits this electromechanical transduction process. In contrast, N-ethylmaleimide and diamide, SH-reagents that only react with exposed SH-groups, showed no effect. These results suggest that one or more reactive SH-groups are present in a functionally important and protected region of the electromechanical transduction protein. Such reactivity can be utilized to identify and characterize this novel membrane motor.

4-Chloromercuribenzenesulfonate↗
PubMed1993

Synthesis of RNA probes by the direct in vitro transcription of PCR-generated DNA templates.

We describe a novel method for the generation of RNA probes based on the direct in vitro transcription of DNA templates amplified by polymerase chain reaction (PCR) using primers with sequence hybrids between the target gene and those of the T7 and T3 RNA polymerases promoters. This method circumvents the need for cloning and allows rapid generation of strand-specific RNA molecules that can be used for the identification of genes in hybridization experiments. We have successfully applied this method to the identification of DNA sequences by Southern blot analysis and library screening.

Base Sequence↗
PubMed1993

Kinesin-mediated vesicular transport in a biochemically defined assay.

Here we have described simple and reproducible methods to observe kinesin-mediated vesicle and microtubule movements under defined conditions using video microscopy. We are optimistic that this assay will provide a useful tool to study kinesin function, regulation, and dynamic physical interactions with membranous organelles and microtubules.

Animals↗
PubMed1993

The structural organization of the pathogenic protozoan Tritrichomonas foetus as seen in replicas of quick frozen, freeze-fractured and deep etched cells.

The quick-freezing and freeze-etching technique was used to analyse the cytoskeleton of Tritrichomonas foetus, a pathogenic protozoan of the urogenital tract of cattle. The cytoplasm presented a network of filamentous structures interacting with each other, with the surface of the hydrogenosomes and the nuclear membrane. Two nm wide filamentous structures were found in the luminal space of the Golgi complex, connecting the two faces of each cisterna. The microtubules of the pelta-axostyle system were connected by bridges 30-40 nm long and 10 nm wide, regularly spaced with an interval of 25 nm. The costa is a structure formed by a complex array of filaments and globous structures. It seems to be connected to the recurrent flagellum through a complex network formed by 15 and 10 nm wide filaments which emerge from the peripheral region of the costa and penetrate into the surface projections of the protozoan body to which the recurrent flagellum is attached. Other filaments were seen connecting the surface of these projections with the surface of the flagellum.

Animals↗
PubMed1992

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.

Animals↗
PubMed1992

Structural domains of the tight junctional intramembrane fibrils.

Freeze-fracture reveals intramembrane fibrils lying along the intermembrane contacts that characterize tight junctions. Tight junctions from a variety of species are reexamined here by rapid freezing prior to freeze-fracture. The tight junction fibril is uprooted alternatively from either the cytoplasmic or the exoplasmic hemibilayer during freeze-cleavage, exposing two distinct but complementary views of its hybrid structure within the same replica. When the transmembrane fibril is uprooted from the exoplasmic hemibilayer it appears on the P-fracture face as a smooth-surfaced cylinder which is sometimes resolved into periodic globular structures. The lack of indication that the P-face cylinder has been pulled out through the opposite membrane half indicates that this domain of the fibril is, in large part, buried in the hydrophobic interior of the membrane. However, when the transmembrane fibril is uprooted from the cytosolic hemibilayer it appears on the E-fracture face as a row of irregular intramembrane particles. The irregular particles on the E-face aspect of the fibril are interpreted as corresponding to transmembrane protein segments that may very well make projections onto the cytosolic surface of the bilayer. En face views of the outermost junction strand between adjacent epithelial cells show periodic lines on the bilayer on each side of the junction which are interpreted as periodic transmembrane protein segments arising from the core structure of the tight junction fibril. If the backbone of the tight junction strand is an inverted cylindrical micelle, it must typically include proteins, which might anchor it to structures outside the membrane bilayer.

Animals↗
PubMed1992

An improved method for the purification of kinesin from bovine adrenal medulla.

A method has been developed for the purification of bovine adrenal kinesin combining ion exchange chromatography on phosphocellulose and Mono-Q (FPLC), affinity binding to microtubules in the presence of tripolyphosphate and gel filtration on Superose 6 (FPLC). From 100 g of tissue this procedure yields 200 micrograms of a remarkably pure kinesin as assayed by SDS-PAGE and electron microscopy of rotary shadowed specimens. The enzyme has a Ca++ ATPase of 0.4 mumol/min per mg and a Mg++ ATPase of 0.03 mumol/min per mg in the absence of microtubules. The addition of microtubules (5 microM) activates the Mg++ ATPase activity by almost 70-fold to a value of 1.9 mumol/min per mg. This purification procedure results in a fairly large amount of a remarkably pure adrenal kinesin with high specific activity which is an important improvement over the method previously available.

Adrenal Medulla↗