Biomedical subjects

D E Koppel

Publications and source records attributed to D E Koppel.

At least 19 recordsLinked to original sources

PubMed2001

Guinea pig fertilin exhibits restricted lateral mobility in epididymal sperm and becomes freely diffusing during capacitation.

The guinea pig sperm protein fertilin functions in sperm-egg plasma membrane binding. Fertilin is initially present in the plasma membrane of the whole head in testicular sperm, then becomes concentrated into the posterior head domain during epididymal passage. Fertilin remains localized to the posterior head plasma membrane following the acrosome reaction, when it functions in sperm-egg interaction. Fluorescence redistribution after photobleaching was used to examine the lateral mobility of fertilin in both acrosome-intact and acrosome-reacted sperm. Fertilin exhibited highly restricted lateral mobility in both testicular and epididymal sperm (D < 10(-10) cm(2)/s). However, fertilin in acrosome-reacted sperm was highly mobile within the membrane bilayer (D = 1.8 x 10(-9) cm(2)/s and %R = 84). Measurement of the lateral mobility of fertilin in capacitated, acrosome-intact sperm revealed two populations of cells. In approximately one-half of the cells, lateral mobility of fertilin was similar to sperm freshly isolated from the cauda epididymis; while in the other half fertilin was highly mobile. The release of fertilin from interactions that restrict its lateral mobility may regulate its function in sperm-egg interaction.

ADAM Proteins↗

PubMed1997

Analysis of the process of localization of fertilin to the sperm posterior head plasma membrane domain during sperm maturation in the epididymis.

Fertilin is a heterodimeric (subunits alpha and beta) sperm plasma membrane protein. Both subunits belong to the ADAM protein family of surface proteins that contain a disintegrin and a metalloprotease domain. Fertilin functions in sperm-egg fusion by binding the sperm to the egg plasma membrane via a binding site in the disintegrin domain of fertilin beta. On testicular sperm of guinea pig, fertilin is distributed on the plasma membrane over the entire sperm head, but is found only on the posterior head once sperm have passed through the epididymis. This redistribution of fertilin to the posterior head can be partially mimicked in vitro if testicular sperm are briefly treated with trypsin. In this study we used immunofluorescence and digital image analysis to analyze how fertilin becomes restricted to the posterior head. We found that fertilin became restricted to the posterior head by migration of anterior head fertilin molecules into the posterior head domain. Comparison of immunofluorescence patterns and immunoblots of fertilin from seven regions of the epididymis showed a temporal correlation between the beginning of fertilin's migration to the posterior head and the proteolytic processing of the full-length fertilin beta precursor (the 85-kDa pro-beta form) to a 75-kDa intermediate, pro-beta*. Completion of the migration coincided with the further cleavage of pro-beta* to the 25- to 28-kDa mature form. Our data suggest that the cleavage of fertilin pro-beta to pro-beta* may initiate fertilin's migration into the posterior head domain and, after localization to that membrane domain, pro-beta* is cleaved to mature beta. We also report evidence that a common mechanism may be used to change the localization pattern of other sperm surface molecules. Other surface proteins were shown to become localized to either the posterior or the anterior head membrane domains on sperm at the same time fertilin became localized to the posterior head. These restrictions of surface protein localizations were also shown to immediately precede the development of the sperm's ability to swim and undergo the acrosome reaction, and thus redistribution of surface proteins may be necessary before sperm become functional.

ADAM Proteins↗

PubMed1997

Barriers to diffusion of plasma membrane proteins form early during guinea pig spermiogenesis.

The plasma membrane of the mature guinea pig sperm is segregated into at least four domains of different composition. Previous studies have shown that some proteins localized within these domains are free to diffuse laterally, suggesting that barriers to protein diffusion are responsible for maintaining the nonuniform distribution of at least some surface proteins in mature sperm. The different membrane domains appear sequentially during sperm morphogenesis in the testis and during later passage through the epididymis. To determine when diffusion barriers become functional during sperm development, we examined the diffusion of two proteins that are expressed on the cell surface of developing spermatids and become segregated to different plasma membrane domains during the course of spermiogenesis. Both proteins exhibited rapid lateral diffusion throughout spermiogenesis, even after they become localized to specific regions of the surface membrane. These results suggest that barriers to membrane diffusion form concomitantly with membrane domains during spermiogenesis.

ADAM Proteins↗

PubMed1996

Analysis of the relationship between the decrease in pH and accumulation of 3-phosphoglyceric acid in developing forespores of Bacillus species.

Analysis of the pH decrease and 3-phosphoglyceric acid (3PGA) accumulation in the forespore compartment of sporulating cells of Bacillus subtilis showed that the pH decrease of 1 to 1.2 units at approximately 4 h of sporulation preceded 3PGA accumulation, as observed previously in B. megaterium. These data, as well as analysis of the forespore pH decrease in asporogenous mutants of B. subtilis, indicated that sigma G-dependent forespore transcription, but not sigma K-dependent mother cell transcription, is required for the forespore pH decrease. Further analysis of these asporogenous mutants showed an excellent correlation between the forespore pH decrease and the forespore's accumulation of 3PGA. These latter results are consistent with our previous suggestion that the decrease in forespore pH results in greatly decreased activity of phosphoglycerate mutase in the forespore, which in turn leads to 3PGA accumulation. In further support of this suggestion, we found that (i) elevating the pH of developing forespores of B. megaterium resulted in rapid utilization of the forespore's 3PGA depot and (ii) increasing forespore levels of PGM approximately 10-fold in B. subtilis resulted in a large decrease in the spore's depot of 3PGA. The B. subtilis strain with a high phosphoglycerate mutase level sporulated, and the spores germinated and went through outgrowth normally, indicating that forespore accumulation of a large 3PGA depot is not essential for these processes.

Acids↗

PubMed1995

Surface expression of the pre-beta subunit of fertilin is regulated at a post-translational level in guinea pig spermatids.

During spermiogenesis in the guinea pig, the spermatid plasma membrane becomes sequentially segregated into three domains of distinct composition. We have previously shown that plasma membrane proteins appear on the cell surface in a temporally regulated manner such that proteins localized to the same domain reach the surface membrane at the same time in sperm development. Fertilin is a cell surface protein restricted to the whole head of testicular sperm; like other proteins restricted to this membrane domain, it does not appear on the cell surface until late (steps 11-13) in spermiogenesis. Using confocal microscopy of immunofluorescently labeled testicular sections, we demonstrate that the pre-beta subunit of fertilin is present in pachytene spermatocytes. It is initially observed in long, strand-like structures that likely represent the endoplasmic reticulum; it later appears in a punctate distribution in the cytoplasm of early spermatids prior to its appearance on the surface membrane in late elongating spermatids. Immunoblotting experiments confirm the presence of the fertilin pre-beta subunit in spermatocytes and early spermatids at the same apparent molecular weight as in later stages. These results suggest that the appearance of fertilin pre-beta subunit on the spermatid surface is regulated by a post-translational mechanism.

ADAM Proteins↗

PubMed1995

Protein translation components are colocalized in granules in oligodendrocytes.

The intracellular distribution of various components of the protein translational machinery was visualized in mouse oligodendrocytes in culture using high resolution fluorescence in situ hybridization and immunofluorescence in conjunction with dual channel confocal laser scanning microscopy. Arginyl-tRNA synthetase, elongation factor 1a, ribosomal RNA, and myelin basic protein mRNA were all co-localized in granules in the processes, veins and membrane sheets of the cell. Colocalization was evaluated by dual channel cross correlation analysis to determine the correlation index (% colocalization) and correlation distance (granule radius), and by single granule ratiometric analysis to determine the distribution of the different components in individual granules. Most granules contained synthetase, elongation factor, ribosomal RNA and myelin basic protein mRNA. These results indicate that several different components of the protein synthetic machinery, including aminoacyl-tRNA synthetases, elongation factors, ribosomes and mRNAs, are colocalized in granules in oligodendrocytes. We propose that these granules are supramolecular complexes containing all of the necessary macromolecular components for protein translation and that they represent a heretofore undescribed subcellular organization of the protein synthetic machinery. This spatial organization may increase the efficiency of protein synthesis and may also provide a vehicle for transport and localization of specific mRNAs within the cell.

Amino Acyl-tRNA Synthetases↗

PubMed1994

Scanning concentration correlation spectroscopy using the confocal laser microscope.

Concentration correlation spectroscopy allows the assessment of molecular motions in complex systems. The technique generally monitors concentration fluctuations by means of some method such as the intensity of fluorescent molecules (fluorescence correlation spectroscopy). We describe here the use of scanning confocal laser microscopy to measure correlation functions in both space and time. This methodology offers two major advantages over conventional methods. First, collecting data from different regions of the sample significantly increases the signal-to-noise ratio. Second, molecular motions of colloidal gold can be analyzed by correlation methods with high temporal and spatial resolution. Using a MRC 600 laser scanning system, we collect data from an ensemble of 768 independent subvolumes and determine the space-time correlation function. We demonstrate the technique using two different types of samples, fluorescently labeled DNA molecules in solution and colloidal gold-tagged lipids in a planar bilayer. This approach, which we term "scanning concentration correlation spectroscopy," provides a straightforward means of performing high resolution correlation analysis of molecular motions with available instrumentation.

Biophysical Phenomena↗

PubMed1994

The internal pH of the forespore compartment of Bacillus megaterium decreases by about 1 pH unit during sporulation.

Previous work has shown that the internal pH of dormant spores of Bacillus species is more than 1 pH U below that of growing cells but rises to that of growing cells in the first minutes of spore germination. In the present work the internal pH of the whole Bacillus megaterium sporangium was measured by the distribution of the weak base methylamine and was found to decrease by approximately 0.4 during sporulation. By using fluorescence ratio image analysis with a fluorescein derivative, 2',7'-bis(2-carboxyethyl)-5 (and -6)-carboxyfluorescein (BCECF), whose fluorescence is pH sensitive, the internal pH of the mother cell was found to remain constant during sporulation at a value of 8.1, similar to that in the vegetative cell. Whereas the internal pH of the forespore was initially approximately 8.1, this value fell to approximately 7.0 approximately 90 min before synthesis of dipicolinic acid and well before accumulation of the depot of 3-phosphoglyceric acid. The pH in the forespore compartment was brought to that of the mother cell by suspending sporulating cells in a pH 8 potassium phosphate buffer plus the ionophore nigericin to clamp the internal pH of the cells to that of the external medium. We suggest that at a minimum, acidification of the forespore may regulate the activity of phosphoglycerate mutase, which is the enzyme known to be regulated to allow 3-phosphoglyceric acid accumulation during sporulation.

Bacillus megaterium↗

PubMed1993

Breaching the diffusion barrier that compartmentalizes the transmembrane glycoprotein CE9 to the posterior-tail plasma membrane domain of the rat spermatozoon.

CE9 is a posterior-tail domain-specific integral plasma membrane glycoprotein of the rat testicular spermatozoon. During epididymal maturation, CE9 undergoes endoproteolytic processing and then redistributes into the anterior-tail plasma membrane domain of the spermatozoon (Petruszak, J. A. M., C. L. Nehme, and J. R. Bartles. 1991. J. Cell. Biol. 114:917-927). We have determined the sequence of CE9 and found it to be a Type Ia transmembrane protein identical to the MRC OX-47 T-cell activation antigen, a member of the immunoglobulin superfamily predicted to have two immunoglobulin-related loops and three asparagine-linked glycans disposed extracellularly. Although encoded by a single gene and mRNA in the rat, the majority of spermatozoal CE9 is of smaller apparent molecular mass than its hepatocytic counterpart due to the under-utilization of sites for asparagine-linked glycosylation. By fluorescence recovery after photobleaching, CE9 was determined to be mobile within the posterior-tail plasma membrane domain of the living rat testicular spermatozoon, thus implying the existence of a regional barrier to lateral diffusion that is presumed to operate at the level of the annulus. Through the development of an in vitro system, the modification of this diffusion barrier to allow for the subsequent redistribution of CE9 into the anterior-tail domain was found to be a time-, temperature-, and energy-dependent process.

Amino Acid Sequence↗

PubMed1991

Migration of the guinea pig sperm membrane protein PH-20 from one localized surface domain to another does not occur by a simple diffusion-trapping mechanism.

The redistribution of membrane proteins on the surface of cells is a prevalent feature of differentiation in a variety of cells. In most cases the mechanism responsible for such redistribution is poorly understood. Two potential mechanisms for the redistribution of surface proteins are: (1) passive diffusion coupled with trapping, and (2) active translocation. We have studied the process of membrane protein redistribution for the PH-20 protein of guinea pig sperm, a surface protein required for sperm binding to the egg zona pellucida (P. Primakoff, H. Hyatt, and D. G. Myles (1985). J. Cell Biol. 101, 2239-2244). PH-20 protein is localized to the posterior head plasma menbrane of the mature sperm cell. Following the exocytotic acrosome reaction, PH-20 protein moves into the newly incorporated inner acrosomal membrane (IAM), placing it in a position favorable for a role in binding sperm to the egg zona pellucida (D. G. Myles, and P. Primakoff (1984), J. Cell Biol. 99, 1634-1641). To analyze the mechanistic basis for this protein migration, we have used fluorescence microscopy and digital image processing to characterize PH-20 protein migration in individual cells. PH-20 protein was observed to move against a concentration gradient in the posterior head plasma membrane. This result argues strongly against a model of passive diffusion followed by trapping in the IAM, and instead suggests that an active process serves to concentrate PH-20 protein toward the boundary separating the posterior head and IAM regions. A transient gradient of PH-20 concentration observed in the IAM suggests that once PH-20 protein reaches the IAM, it is freely diffusing. Additionally, we observed that migration of PH-20 protein was calcium dependent.

Acrosome↗

PubMed1991

Condensation of the forespore nucleoid early in sporulation of Bacillus species.

Fluorescence microscopic examination coupled with digital videoimage analysis of 4',6-diamidino-2-phenylindole-stained sporulating cells of Bacillus megaterium or Bacillus subtilis revealed a striking condensation of the forespore nucleoid. While both mother cell and forespore compartments had equal amounts of DNA, the forespore nucleoid became greater than 2-fold more condensed than the mother cell nucleoid. The condensation of the forespore nucleoid began after only the first hour of sporulation, 2 to 3 h before expression of most forespore-specific genes including those for small, acid-soluble spore proteins, and was abolished in spo0 mutants but not in spoII or spoIII mutants. It is possible that this striking condensation of forespore DNA plays some role in modulating gene expression during sporulation.

Bacillus megaterium↗

PubMed1990

Normal band 3-cytoskeletal interactions are maintained on tanktreading erythrocytes.

Normal nonnucleated erythrocytes subjected to continuous hydrodynamic shear exhibit membrane deformation or "tanktreading," a process important for reduction of the bulk viscosity of circulating blood. To characterize the effect of this unique process on the erythrocyte membrane we have measured the lateral diffusion of band 3 during tanktreading. Band 3 is normally constrained through interactions with the spectrin-actin cytoskeleton, therefore, any significant disruption of these interactions would result in alterations in band 3 dynamics. Band 3 of human erythrocytes was labeled with dichlorotriazinyl amino fluorescein. After laser photobleaching of an equatorial stripe, fluorescence images were recorded from cells in the presence or absence of shear. The amplitude of induced nonuniformity in the surface distribution of fluorescence was calculated directly from images of unsheared cells. In shear the bleached line rotated with the tanktreading motion of the cells. The surface integral of fluorescence oscillated with this motion. For this case, the amplitude of photobleaching-induced nonuniformity was defined as the amplitude at the fundamental frequency of fast Fourier transforms in time of the oscillations. Shear stress-induced membrane flow did not interrupt the linkage of band 3 with the erythrocyte cytoskeleton. Diffusion coefficient and mobile fraction (1.5 +/- 0.5 x 10(-10) cm2/s and 54 +/- 11%, respectively) were unaffected by shear. The rate of fluorescence recovery of cells in shear was also similar at the centers and at the edges, where in-plane shear forces are maximal.

Anion Exchange Protein 1, Erythrocyte↗

PubMed1990

Evidence that proteolysis of the surface is an initial step in the mechanism of formation of sperm cell surface domains.

On terminally differentiated sperm cells, surface proteins are segregated into distinct surface domains that include the anterior and posterior head domains. We have analyzed the formation of the anterior and posterior head domains of guinea pig sperm in terms of both the timing of protein localization and the mechanism(s) responsible. On testicular sperm, the surface proteins PH-20, PH-30 and AH-50 were found to be present on the whole cell (PH-20) or whole head surface (PH-30, AH-50). On sperm that have completed differentiation (cauda epididymal sperm), PH-20 and PH-30 proteins were restricted to the posterior head domain and AH-50 was restricted to the anterior head domain. Thus these proteins become restricted in their distribution late in sperm differentiation, after sperm leave the testis. We discovered that the differentiation process that localizes these proteins can be mimicked in vitro by treating testicular sperm with trypsin. After testicular sperm were treated with 20 micrograms/ml trypsin for 5 min at room temperature, PH-20, PH-30, and AH-50 were found localized to the same domains to which they are restricted during in vivo differentiation. The in vitro trypsin-induced localization of PH-20 to the posterior head mimicked the in vivo differentiation process quantitatively as well as qualitatively. The quantitative analysis showed the process of PH-20 localization involves the migration of surface PH-20 from other regions to the posterior head domain. Immunoprecipitation experiments confirmed that there is protease action in vivo on the sperm surface during the late stages of sperm differentiation. Both the PH-20 and PH-30 proteins were shown to be proteolytically cleaved late in sperm differentiation. These findings strongly implicate proteolysis of surface molecules as an initial step in the mechanism of formation of sperm head surface domains.

Animals↗

PubMed1989

Analysis of heterogeneous fluorescence photobleaching by video kinetics imaging: the method of cumulants.

The method of cumulants has been applied to digital video fluorescence microscopy. The method is used to reconstruct the distribution of fluorescent molecules before the initiation of fluorescence photobleaching, and to characterize heterogeneous photobleaching by imaging one or more of the cumulants of the bleaching decay rate. Using the pipelined pixel processor of the image analysis system for the bulk of the calculations, rather than the general-purpose host-computer CPU, the video kinetics imaging can be performed in near real-time. The method is applied to chick embryo myotubes labelled with fluorescein-conjugated alpha-bungarotoxin. The pre-bleach fluorescence distribution is derived, and the image of fluorescein fluorescence is separated from glutaraldehyde-induced autofluorescence on the basis of the spatially resolved average photobleaching decay rate.

Animals↗

PubMed1988

Restricted lateral diffusion of PH-20, a PI-anchored sperm membrane protein.

The rate of lateral diffusion of integral membrane proteins is constrained in cells, but the constraining factors for most membrane proteins have not been defined. PH-20, a sperm surface protein involved in sperm-egg adhesion, was shown to be anchored in the plasma membrane by attachment to the lipid phosphatidylinositol and to have a diffusion rate that is highly restricted on testicular sperm, being more than a thousand times slower than lipid diffusion. These results support the hypothesis that lateral mobility of a membrane protein can be regulated exclusively by interactions of its ectodomain.

Animals↗

PubMed1987

Diffusion of dihydropyridine calcium channel antagonists in cardiac sarcolemmal lipid multibilayers.

A membrane bilayer pathway model has been proposed for the interaction of dihydropyridine (DHP) calcium channel antagonists with receptors in cardiac sarcolemma (Rhodes, D.G., J.G. Sarmiento, and L.G. Herbette. 1985. Mol. Pharmacol. 27:612-623) involving drug partition into the bilayer with subsequent receptor binding mediated (though probably not rate-limited) by diffusion within the bilayer. Recently, we have characterized the partition step, demonstrating that DHPs reside, on a time-average basis, near the bilayer hydrocarbon core/water interface. Drug distribution about this interface may define a plane of local concentration for lateral diffusion within the membrane. The studies presented herein examine the diffusional dynamics of an active rhodamine-labeled DHP and a fluorescent phospholipid analogue (DiIC16) in pure cardiac sarcolemmal lipid multibilayer preparations as a function of bilayer hydration. At maximal bilayer hydration, the drug diffuses over macroscopic distances within the bilayer at a rate identical to that of DiI (D = 3.8 X 10(-8) cm2/s), demonstrating the overall feasibility of the membrane diffusion model. The diffusion coefficients for both drug and lipid decreased substantially as the bilayers were dehydrated. While identical at maximal hydration, drug diffusion was significantly slower than that of DiIC16 in partially dehydrated bilayers, probably reflecting differences in mass distribution of these probes in the bilayer.

Animals↗

PubMed1987

Lateral diffusion of the PH-20 protein on guinea pig sperm: evidence that barriers to diffusion maintain plasma membrane domains in mammalian sperm.

PH-20 protein on the plasma membrane (PH-20PM) is restricted to the posterior head of acrosome-intact guinea pig sperm. During the exocytotic acrosome reaction the inner acrosomal membrane (IAM) becomes continuous with the posterior head plasma membrane, and PH-20PM migrates to the IAM. There it joins a second population of PH-20 protein localized to this region of the acrosomal membrane (PH-20AM) (Cowan, A.E., P. Primakoff, and D.G. Myles, 1986, J. Cell Biol. 103:1289-1297). To investigate how the localized distributions of PH-20 protein are maintained, the lateral mobility of PH-20 protein on these different membrane domains was determined using fluorescence redistribution after photobleaching. PH-20PM on the posterior head of acrosome-intact sperm was found to be mobile, with a diffusion coefficient and percent recovery typical of integral membrane proteins (D = 1.8 X 10(-10) cm2/s; %R = 73). This value of D was some 50-fold lower than that found for the lipid probe 1,1-ditetradecyl 3,3,3',3'-tetramethylindocarbocyanine perchlorate (C14diI) in the same region (D = 8.9 X 10(-9) cm2/s). After migration to the IAM of acrosome-reacted sperm, this same population of molecules (PH-20PM) exhibited a 30-fold increase in diffusion rate (D = 4.9 X 10(-9) cm2/s; %R = 78). This rate was similar to diffusion of the lipid probe C14diI in the IAM (D = 5.4 X 10(-9) cm2/s). The finding of free diffusion of PH-20PM in the IAM of acrosome-reacted sperm supports the proposal that PH-20 is maintained within the IAM by a barrier to diffusion at the domain boundary. The slower diffusion of PH-20PM on the posterior head of acrosome-intact sperm is also consistent with localization by barriers to diffusion, but does not rule out alternative mechanisms.

Acrosome↗

PubMed1987

High lateral mobility of endogenous and transfected alkaline phosphatase: a phosphatidylinositol-anchored membrane protein.

The lateral mobility of alkaline phosphatase (AP) in the plasma membrane of osteoblastic and nonosteoblastic cells was estimated by fluorescence redistribution after photobleaching in embryonic and in tumor cells, in cells that express AP naturally, and in cells transfected with an expression vector containing AP cDNA. The diffusion coefficient (D) and the mobile fraction, estimated from the percent recovery (%R), were found to be cell-type dependent ranging from (0.58 +/- 0.16) X 10(-9) cm2s-1 and 73.3 +/- 10.5 in rat osteosarcoma cells ROS 17/2.8 to (1.77 +/- 0.51) X 10(-9) cm2s-1 and 82.8 +/- 2.5 in rat osteosarcoma cells UMR106. Similar values of D greater than or equal to 10(-9) cm2s-1 with approximately 80% recovery were also found in fetal rat calvaria cells, transfected skin fibroblasts, and transfected AP-negative osteosarcoma cells ROS 25/1. These values of D are many times greater than "typical" values for membrane proteins, coming close to those of membrane lipid in fetal rat calvaria and ROS 17/2.8 cells (D = [4(-5)] X 10(-9) cm2s-1 with 75-80% recovery), estimated with the hexadecanoyl aminofluorescein probe. In all cell types, phosphatidylinositol (PI)-specific phospholipase C released 60-90% of native and transfection-expressed AP, demonstrating that, as in other tissue types, AP in these cells is anchored in the membrane via a linkage to PI. These results indicate that the transfected cells used in this study possess the machinery for AP insertion into the membrane and its binding to PI. The fast AP mobility appears to be an intrinsic property of the way the protein is anchored in the membrane, a conclusion with general implications for the understanding of the slow diffusion of other membrane proteins.

Alkaline Phosphatase↗