Patterns of kinship in groups of free-living sperm whales (Physeter macrocephalus) revealed by multiple molecular genetic analyses.

Mature female sperm whales (Physeter macrocephalus) live in socially cohesive groups of 10-30, which include immature animals of both sexes, and within which there is communal care of the young. We examined kinship in such groups using analyses of microsatellite DNA, mitochondrial DNA sequence, and sex-linked markers on samples of sloughed skin collected noninvasively from animals in three groups off the coast of Ecuador. Social groups were defined through photographic identification of individuals. Each group contained about 26 members, mostly female (79%). Relatedness was greater within groups, as compared to between groups. Particular mitochondrial haplotypes were characteristic of groups, but all groups contained more than one haplotype. The data are generally consistent with each group being comprised of several matrillines from which males disperse at about the age of 6 years. There are indications of paternal relatedness among grouped individuals with different mitochondrial haplotypes, suggesting long-term associations between different matrilines.

Internal molecular motions of bacteriorhodopsin: hydration-induced flexibility studied by quasielastic incoherent neutron scattering using oriented purple membranes.

Quasielastic incoherent neutron scattering from hydrogen atoms, which are distributed nearly homogeneously in biological molecules, allows the investigation of diffusive motions occurring on the pico- to nanosecond time scale. A quasielastic incoherent neutron scattering study was performed on the integral membrane protein bacteriorhodopsin (BR), which is a light-driven proton pump in Halobacterium salinarium. BR is embedded in lipids, forming patches in the cell membrane of the organism, which are the so called purple membranes (PMs). Measurements were carried out at room temperature on oriented PM-stacks hydrated at two different levels (low hydration, h = 0.03 g of D2O per g of PM; high hydration, h = 0.28 g of D2O per g of PM) using time-of-flight spectrometers. From the measured spectra, different diffusive components were identified and analyzed with respect to the influence of hydration. This study supports the idea that a decrease in hydration results in an appreciable decrease in internal molecular flexibility of the protein structure. Because it is known from studies on the function of BR that the pump activity is reduced if the hydration level of the protein is insufficient, we conclude that the observed diffusive motions are essential for the function of this protein. A detailed analysis and classification of the different kinds of diffusive motions, predominantly occurring in PMs under physiological conditions, is presented.

Phenotypic variations among paternal centrosomes expressed within the zygote as disparate microtubule lengths and sperm aster organization: correlations between centrosome activity and developmental success.

This study describes a paternal effect on sperm aster size and microtubule organization during bovine fertilization. Immunocytochemistry using tubulin antibodies quantitated with confocal microscopy was used to measure the diameter of the sperm aster and assign a score (0-3) based on the degree of radial organization (0, least organized; 3, most organized). Three bulls (A-C) were chosen based on varying fertility (A, lowest fertility; C, highest fertility) as assessed by nonreturn to estrus after artificial insemination and in vitro embryonic development to the blastocyst stage. The results indicate a statistically significant bull-dependent difference in diameter of the sperm aster and in the organization of the sperm astral microtubules. Insemination from bull A resulted in an average sperm aster diameter of 101.4 microm (76.3% of oocyte diameter). This significantly differs (P < or = 0.0001) from the average sperm aster diameters produced after inseminations from bull B (78.2 microm; 60.8%) or bull C (77.9 microm; 57.8%), which themselves displayed no significant differences. The degree of radial organization of the sperm aster was also bull-dependent. Sperm asters organized by bull A-derived sperm had an average quality score of 1.8, which was higher than that of bull B (1.4; P < or = 0.0005) or bull C (1.2; P < or = 0.0001). Results with bulls B and C were also significantly different (P < or = 0.025). These results indicate that the paternally derived portion of the centrosome varies among males and that this variation affects male fertility, the outcome of early development, and, therefore, reproductive success.

Z-membranes: artificial organelles for overexpressing recombinant integral membrane proteins.

We have expressed a fusion protein formed between the avian infectious bronchitis virus M protein and the bacterial enzyme beta-glucuronidase in transgenic tobacco cells. Electron microscope images of such cells demonstrate that overexpression of this fusion protein gives rise to a type of endoplasmic reticulum membrane domain in which adjacent membranes become zippered together apparently as a consequence of the oligomerizing action of beta-glucuronidase. These zippered (Z-) membranes lack markers of the endoplasmic reticulum (NADH cytochrome c reductase and ribosomes) and accumulate in the cells in the form of multilayered scroll-like structures (up to 2 micrometers in diameter; 20-50 per cell) without affecting plant growth. The discovery of Z-membranes has broad implications for biology and biotechnology in that they provide a means for accumulating large quantities of recombinant membrane proteins within discrete domains of native membranes.

Sperm capacitation in humans is transient and correlates with chemotactic responsiveness to follicular factors.

In humans, only a small fraction (2-12%) of a sperm population can respond by chemoattraction to follicular factors. This recent finding led to the hypothesis that chemotaxis provides a mechanism for selective recruitment of functionally mature spermatozoa (i.e., of capacitated spermatozoa, which possess the potential to undergo the acrosome reaction and fertilize the egg). This study aimed to examine this possibility. Capacitated spermatozoa were identified by their ability to undergo the acrosome reaction upon stimulation with phorbol 12-myristate 13-acetate. Under capacitating conditions, only a small portion (2-14%) of the spermatozoa were found to be capacitated. The spermatozoa were then separated according to their chemotactic activity, which resulted in a subpopulation enriched with chemotactically responsive spermatozoa and a subpopulation depleted of such spermatozoa. The level of capacitated spermatozoa in the former was approximately 13-fold higher than that in the latter. The capacitated state was temporary (50 min < life span < 240 min), and it was synchronous with the chemotactic activity. A continuous process of replacement of capacitated/chemotactic spermatozoa within a sperm population was observed. Spermatozoa that had stopped being capacitated did not become capacitated again, which indicates that the capacitated state is acquired only once in a sperm's lifetime. A total sperm population depleted of capacitated spermatozoa stopped being chemotactic. When capacitated spermatozoa reappeared, chemotactic activity was restored. These observations suggest that spermatozoa acquire their chemotactic responsiveness as part of the capacitation process and lose this responsiveness when the capacitated state is terminated. We suggest that the role of sperm chemotaxis in sperm-egg interaction in vivo may indeed be selective recruitment of capacitated spermatozoa for fertilizing the egg.

Delayed male maturity is a cost of producing large sperm in Drosophila.

Among fruit-fly species of the genus Drosophila there is remarkable variation in sperm length, with some species producing gigantic sperm (e.g., > 10 times total male body length). These flies are also unusual in that males of some species exhibit a prolonged adult nonreproductive phase. We document sperm length, body size, and sex-specific ages of reproductive maturity for 42 species of Drosophila and, after controlling for phylogeny, test hypotheses to explain the variation in rates of sexual maturation. Results suggest that delayed male maturity is a cost of producing long sperm. A possible physiological mechanism to explain the observed relationship is discussed.

The intrinsic ability of ribosomes to bind to endoplasmic reticulum membranes is regulated by signal recognition particle and nascent-polypeptide-associated complex.

Signal peptides direct the cotranslational targeting of nascent polypeptides to the endoplasmic reticulum (ER). It is currently believed that the signal recognition particle (SRP) mediates this targeting by first binding to signal peptides and then by directing the ribosome/nascent chain/SRP complex to the SRP receptor at the ER. We show that ribosomes can mediate targeting by directly binding to translocation sites. When purified away from cytosolic factors, including SRP and nascent-polypeptide-associated complex (NAC), in vitro assembled translation intermediates representing ribosome/nascent-chain complexes efficiently bound to microsomal membranes, and their nascent polypeptides could subsequently be efficiently translocated. Because removal of cytosolic factors from the ribosome/nascent-chain complexes also resulted in mistargeting of signalless nascent polypeptides, we previously investigated whether readdition of cytosolic factors, such as NAC and SRP, could restore fidelity to targeting. Without SRP, NAC prevented all nascent-chain-containing ribosomes from binding to the ER membrane. Furthermore, SRP prevented NAC from blocking ribosome-membrane association only when the nascent polypeptide contained a signal. Thus, NAC is a global ribosome-binding prevention factor regulated in activity by signal-peptide-directed SRP binding. A model presents ribosomes as the targeting vectors for delivering nascent polypeptides to translocation sites. In conjunction with signal peptides, SRP and NAC contribute to this specificity of ribosomal function by regulating exposure of a ribosomal membrane attachment site that binds to receptors in the ER membrane.

Phospholipase D is present on Golgi-enriched membranes and its activation by ADP ribosylation factor is sensitive to brefeldin A.

ADP ribosylation factor (ARF) is a small guanosine triphosphate (GTP)-binding protein that regulates the binding of coat proteins to membranes and is required for several stages of vesicular transport. ARF also stimulates phospholipase D (PLD) activity, which can alter the lipid content of membranes by conversion of phospholipids into phosphatidic acid. Abundant PLD activity was found in Golgi-enriched membranes from several cell lines. Golgi PLD activity was greatly stimulated by ARF and GTP analogs and this stimulation could be inhibited by brefeldin A (BFA), a drug that blocks binding of ARF to Golgi membranes. Furthermore, in Golgi membranes from BFA-resistant PtK1 cells, basal PLD activity was high and not stimulated by exogenous ARF or GTP analogs. Thus, ARF activates PLD on the Golgi complex, suggesting a possible link between transport events and the underlying architecture of the lipid bilayer.

Extraordinary divergence and positive Darwinian selection in a fusagenic protein coating the acrosomal process of abalone spermatozoa.

During fertilization in marine invertebrates, fusion between sperm and egg cell membranes occurs at the tip of the sperm acrosomal process. In abalone sperm the acrosomal process is coated with an 18-kDa protein. In situ, this protein has no effect on the egg vitelline envelope, but in vitro it is a potent fusagen of liposomes. Thus, the 18-kDa protein may mediate membrane fusion between the gametes, a step in gamete recognition known to restrict heterospecific fertilization in other species. The cDNA and deduced amino acid sequences of the 18-kDa protein were determined for five species of California abalone. The deduced amino acid sequences exhibit extraordinary divergence; the percent identity varies from 27% to 87%. Analysis of nucleotide substitution shows extremely high frequencies of amino acid-altering substitution compared to silent substitution, demonstrating that positive Darwinian selection promotes the divergence of this protein. However, amino acid replacement is conservative with respect to size and polarity of residue. The data support the developing idea that in free-spawning marine invertebrates, the proteins mediating fertilization may be subjected to intense, and as yet unknown, selective forces. The extraordinary divergence of fertilization proteins may be related to the establishment of barriers to heterospecific fertilization.

Incorporation of acetylcholine receptors and Cl- channels in Xenopus oocytes injected with Torpedo electroplaque membranes.

A method was developed to transplant assembled nicotinic acetylcholine receptors (AcChoRs) and Cl- channels from the electric organ of Torpedo to the membrane of Xenopus oocytes. Membrane vesicles from Torpedo electroplaques were injected into the oocytes and, within a few hours, the oocyte membrane acquired AcChoRs and Cl- channels. The mechanism of expression of these receptors and channels is very different from that which follows the injection of mRNA, since the appearance of receptors after membrane injection does not require de novo protein synthesis or N-glycosylation. This, and other controls, indicate that the foreign receptor-bearing membranes fuse with the oocyte membrane and cause the appearance of functional receptors and channels. All this makes the Xenopus oocyte an even more powerful tool for studies of the structure and function of membrane proteins.

Nonneuronal expression of Rab3A: induction during adipogenesis and association with different intracellular membranes than Rab3D.

Rab3A is a small GTP-binding protein expressed predominantly in brain and neuroendocrine cells, in which it is associated with synaptic and synaptic-like vesicles, respectively. Here we report that adult mouse fat cells and 3T3-L1 adipocytes also express Rab3A mRNA and protein. They do not express synaptophysin, an abundant protein in synaptic vesicles or synaptic-like vesicles. The amount of Rab3A mRNA and protein, like that of the highly homologous isoform Rab3D, increases severalfold during differentiation of 3T3-L1 fibroblasts into mature adipocytes. In fat cells, most Rab3D and Rab3A protein is bound to membrane, irrespective of insulin addition. Rab3A and Rab3D are localized in different subcellular compartments, since about half of the Rab3A, but none of the Rab3D, is associated with a low-density organelle(s). Rab3D and Rab3A may be involved in different pathways of regulated exocytosis in adipocytes. Moreover, in adipocytes Rab3A may define an exocytic organelle that is different from synaptic vesicles or synaptic-like microvesicles found in neuronal and endocrine cells.

Immunolocalization of the mercurial-insensitive water channel and glycerol intrinsic protein in epithelial cell plasma membranes.

Two water channel homologs were cloned recently from rat kidney, mercurial-insensitive water channel (MIWC) and glycerol intrinsic protein (GLIP). Polyclonal antibodies were raised against synthetic C-terminal peptides and purified by affinity chromatography. MIWC and GLIP antibodies recognized proteins in rat kidney with an apparent molecular mass of 30 and 27 kDa, respectively, and did not cross-react. By immunofluorescence, MIWC and GLIP were expressed together on the basolateral plasma membrane of collecting duct principal cells in kidney. By immunohistochemistry, MIWC and GLIP were expressed on tracheal epithelial cells with greater expression of GLIP on the basal plasma membrane and MIWC on the lateral membrane; only MIWC was expressed in bronchial epithelia. In eye, GLIP was expressed in conjunctival epithelium, whereas MIWC was found in iris, ciliary body, and neural cell layers in retina. MIWC and GLIP colocalized on the basolateral membrane of villus epithelial cells in colon and brain ependymal cells. Expression of MIWC and GLIP was not detected in small intestine, liver, spleen, endothelia, and cells that express water channels CHIP28 or WCH-CD. These studies suggest water/solute transporting roles for MIWC and GLIP in the urinary concentrating mechanism, cerebrospinal fluid absorption, ocular fluid balance, fecal dehydration, and airway humidification. The unexpected membrane colocalization of MIWC and GLIP in several tissues suggests an interaction at the molecular and/or functional levels.

A chloroplast homologue of the signal recognition particle subunit SRP54 is involved in the posttranslational integration of a protein into thylakoid membranes.

The mechanisms involved in the integration of proteins into the thylakoid membrane are largely unknown. However, many of the steps of this process for the light-harvesting chlorophyll a/b protein (LHCP) have been described and reconstituted in vitro. LHCP is synthesized as a precursor in the cytosol and posttranslationally imported into chloroplasts. Upon translocation across the envelope membranes, the N-terminal transit peptide is cleaved, and the apoprotein is assembled into a soluble "transit complex" and then integrated into the thylakoid membrane via three transmembrane helices. Here we show that 54CP, a chloroplast homologue of the 54-kDa subunit of the mammalian signal recognition particle (SRP54), is essential for transit complex formation, is present in the complex, and is required for LHCP integration into the thylakoid membrane. Our data indicate that 54CP functions posttranslationally as a molecular chaperone and potentially pilots LHCP to the thylakoids. These results demonstrate that one of several pathways for protein routing to the thylakoids is homologous to the SRP pathway and point to a common evolutionary origin for the protein transport systems of the endoplasmic reticulum and the thylakoid membrane.