PubMed Health⌕ Search

Biomedical subjects

A E Cowan

Publications and source records attributed to A E Cowan.

12 recordsLinked to original sources
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↗
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

Biogenesis of surface domains during spermiogenesis in the guinea pig.

During mammalian spermiogenesis a spherical spermatid is transformed into a highly asymmetric sperm cell. Concurrently, the plasma membrane of the cell develops into a mosaic of discrete membrane regions, with each region containing a unique set of proteins. Biogenesis of these surface domains was studied by following the surface expression and localization of nine different antigens during spermiogenesis. Each of these antigens exhibits one of four distinct patterns of localization on testicular sperm (whole cell, whole head, anterior tail, and posterior tail), indicating that there are at least three distinct surface domains on testicular sperm. Our results on the timing of antigen localization suggest that the generation of surface domains in mammalian sperm is a complex process. This process involves temporal and spatial regulation of surface expression of the antigens, as well as the specific removal of antigens from inappropriate domains after they have reached the cell surface.

Animals↗
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↗
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

Rearrangement of sperm surface antigens prior to fertilization.

During spermiogenesis and epididymal transit, proteins on the sperm surface become localized to specific domains. In at least one case (PH-20), the protein is initially inserted throughout the membrane and subsequently becomes restricted to a domain by some mechanism that has not yet been determined. Other proteins could become localized through localized insertion. The sperm surface is a dynamic structure that is altered even after the spermatozoon leaves the male. In the female reproductive tract the spermatozoa undergo capacitation and the acrosome reaction that enables them to fertilize the egg. Both of these processes are accompanied by alterations in protein localization: the PT-1 protein migrates during capacitation, and the PH-20 protein migrates after the acrosome reaction. In addition, an upregulation of the surface expression of PH-20 occurs during the acrosome reaction. This additional PH-20 is incorporated into the plasma membrane by the irreversible fusion of the acrosomal membrane with the plasma membrane. The acrosomal membrane contains PH-20 protein that has been stored there since the formation of the acrosome at the spermatid stage of spermiogenesis. Proteins that are freely diffusing must be maintained in a domain by a mechanism that does not involve immobilization or slowing of protein diffusion. We have suggested that barriers to membrane protein diffusion exist at the equatorial region, the posterior ring, and the annulus and that they are responsible for maintaining a localized distribution of at least some of the surface proteins. The migration of surface proteins could result from an alteration of these barriers, a change in the protein structure so that it can pass through the barrier, or active transport across the barrier. These observed changes in surface expression (localization and the level of expression) may be acting to control surface function post-testicularly.

Acrosome↗
PubMed1986

Sperm exocytosis increases the amount of PH-20 antigen on the surface of guinea pig sperm.

Evidence has been presented that the PH-20 protein functions in sperm adhesion to the egg zona pellucida (Primakoff, P., H. Hyatt, and D. G. Myles, 1985, J. Cell Biol., 101:2239-2244). The PH-20 protein migrates from its original surface domain to a new surface domain after the acrosome reaction (Myles, D. G., and P. Primakoff, 1984, J. Cell Biol., 99:1634-1641). The acrosome reaction is an exocytotic event that results in insertion of a region of the secretory granule membrane, the inner acrosomal membrane (IAM), into the plasma membrane. After the acrosome reaction, PH-20 protein migrates to the IAM from its initial domain on the posterior head surface. We have now found a new dynamic feature of the regulation of PH-20 protein on the sperm surface; exocytosis increases the surface expression of PH-20 protein. After the acrosome reaction there is an approximately threefold increase in the number of PH-20 antigenic sites on the sperm surface. These new antigenic sites are revealed on the surface by insertion of the IAM into the plasma membrane. Our evidence indicates that before the acrosome reaction an intracellular population of PH-20 antigen is localized to the IAM. When migration of the surface population of the PH-20 protein is prevented, PH-20 protein can still be detected on the IAM of acrosome-reacted sperm. Also, PH-20 protein can be detected on the IAM of permeabilized acrosome-intact sperm by indirect immunofluorescence. Thus, the sperm cell regulates the amount of PH-20 protein on its surface by sequestering about two-thirds of the protein on an intracellular membrane and subsequently exposing this population on the cell surface by an exocytotic event. This may be a general mechanism for regulating cell surface composition where a rapid increase in the amount of a cell surface protein is required.

Acrosome↗
PubMed1985

Mapping the distribution of differentiation potential for intestine, muscle, and hypodermis during early development in Caenorhabditis elegans.

We have analyzed the differentiation potential of cells in early embryos of Caenorhabditis elegans by assessing the production of markers for intestinal, muscle, and hypodermal cell differentiation in cleavage-arrested blastomeres. Our results show that differentiation potential does not always segregate during cleavage in a linear fashion, i.e., a blastomere can express a differentiation potential that is absent in its parent blastomere and vice versa. Furthermore, the expression of a particular differentiation program by certain cleavage-arrested blastomeres is an exclusive event in that each cell will express only one program of differentiation, even though it may have the potential to express several.

Animals↗