Elizabeth A. Howes

Extracellular signal-regulated kinases 1/2 are serum-stimulated "Bim(EL) kinases" that bind to the BH3-only protein Bim(EL) causing its phosphorylation and turnover.

Bim, a “BH3-only” protein, is expressed de novo following withdrawal of serum survival factors and promotes cell death. We have shown previously that activation of the ERK1/2 pathway promotes phosphorylation of BimEL, targeting it for degradation via the proteasome. However, the nature of the kinase responsible for BimEL phosphorylation remained unclear. We now show that BimEL is phosphorylated on at least three sites in response to activation of the ERK1/2 pathway. By using the peptidylprolyl isomerase, Pin1, as a probe for proline-directed phosphorylation, we show that ERK1/2-dependent phosphorylation of BimEL occurs at (S/T)P motifs. ERK1/2 phosphorylates BimEL, but not BimS or BimL, in vitro, and mutation of Ser65 to alanine blocks the phosphorylation of BimEL by ERK1/2 in vitro and in vivo and prevents the degradation of the protein following activation of the ERK1/2 pathway. We also find that ERK1/2, but not JNK, can physically associate with GST-BimEL, but not GST-BimL or GST-BimS, in vitro. ERK1/2 also binds to full-length BimEL in vivo, and we have localized a potential ERK1/2 “docking domain” lying within a 27-amino acid stretch of the BimEL protein. Our findings provide new insights into the post-translational regulation of BimEL and the role of the ERK1/2 pathway in cell survival signaling.

Cholesterol Efflux Alters Lipid Raft Stability and Distribution During Capacitation of Boar Spermatozoa

Abstract A reduction in plasma membrane cholesterol is one of the early events that either triggers or is closely associated with capacitation of mammalian spermatozoa. In this investigation, we have examined the effects of cholesterol efflux on tyrosine phosphorylation, lipid diffusion, and raft organization in boar spermatozoa. Results show that a low level of cholesterol efflux, mediated by 5 mM methyl-β-cyclodextrin (MBCD), enhances capacitation and induces phosphorylation of two proteins at 26 and 15 kDa without affecting sperm viability. Lipid diffusion rates under these conditions are largely unaffected except when cholesterol efflux is excessive. Low-density Triton X100-insoluble complexes (lipid rafts) were isolated from spermatozoa and found to have a restricted profile of proteins. Capacitation-associated cholesterol efflux has no effect on raft composition, but cholesterol depletion destabilizes them completely and phosphorylation is suppressed. During MBCD-mediated capacitation, the distribution of GM1 gangliosides on spermatozoa changes in a sequential manner from overlying the sperm tail to clustering on the sperm head. It is concluded that there is a safe window for removal of plasma membrane cholesterol from spermatozoa within which protein phosphorylation and polarized migration of lipid rafts take place. A preferential loss of cholesterol from the nonraft pool may be the stimulus that promotes raft clustering over the anterior sperm head.

Expression of a testis-specific putative actin-capping protein associated with the developing acrosome during rat spermiogenesis

Actin‐capping proteins are ubiquitous components of mammalian cells. They are known to regulate the polymerization state of actin and hence indirectly control the activity of the cytoskeleton and cell shape. As part of our investigation into the molecular mechanisms that direct differentiation of a round spermatid into an elongating spermatozoa, we report on a testis‐specific 1.7‐kb transcript from rat testis with sequence similarities to the α subunit of actin‐capping proteins (ACPs) from somatic cells. The transcript contains a putative cAMP‐responsive motif (CREM) upstream of the initiation codon in the DNA sequence and is expressed postmeiotically, first appearing between 20 and 30 days of postnatal development. The primary amino acid sequence is 90% identical to that of a previously identified testis‐specific mouse protein, gsg3, both showing approximately 40% homology to the α subunit of somatic ACPs. An affinity‐purified polyclonal antibody to a synthetic peptide derived from the rat transcript identified a 32‐kDa protein on Western blots of testicular extracts. Indirect immunofluorescent localization of the protein on frozen sections of adult rat testis showed that it is intracellular and accumulates asymmetrically in the cytoplasm of round spermatids coincident with the position of the developing acrosome. This spatial expression parallels the distribution of F‐actin during sperm differentiation, supporting the hypothesis that testis‐specific ACPs have an important role in determining the final shape of mature sperm heads. A disturbance in the expression of these ACPs may underlie many of the abnormalities in sperm morphology observed in infertile semen. Mol. Reprod. Dev. 49:81–91, 1998.

Tracking diffusion of GM1 gangliosides and zona pellucida binding molecules in sperm plasma membranes following cholesterol efflux

The molecules on mammalian spermatozoa that mediate recognition and binding to the zona pellucida of the egg are still not understood. Current concepts favour their assembly into multimolecular complexes in the plasma membrane in response to cholesterol efflux, an important step during sperm capacitation. Here, we track in real time diffusion of cross-linked clusters containing zona-binding molecules and GM1 gangliosides in the plasma membrane of live boar spermatozoa before and after cholesterol reduction. Both GM1 gangliosides and zona-binding molecules partition into a low density Triton X100 resistant phase suggesting their association with lipid rafts. Initially, GM1 and zona-binding molecules localize to the apical ridge on the acrosome but following cholesterol efflux with methyl-beta-cyclodextrin, clusters containing zona-binding molecules diffuse randomly over the acrosomal domain. Diffusing clusters of either type do not access the postacrosome. Spermatozoa agglutinated head-to-head show contact-induced coalescence of GM1 gangliosides (but not zona-binding molecules) suggestive of a specific mechanosensitive response. Thus, cholesterol efflux initiates diffusion (and possibly formation) of novel lipid raft-like structures containing zona-binding molecules over the sperm acrosome. We hypothesise that in combination with contact coalescence, these mechanisms concentrate important molecules to the appropriate site on the sperm surface to mediate zona binding.

The Equatorial Subsegment in Mammalian Spermatozoa Is Enriched in Tyrosine Phosphorylated Proteins

Abstract The equatorial subsegment (EqSS) was originally identified by atomic force microscopy as a discrete region within the equatorial segment of Artiodactyl spermatozoa. In this investigation, we show that the EqSS is enriched in tyrosine phosphorylated proteins and present preliminary evidence for its presence in mouse and rat spermatozoa. The anti-phosphotyrosine monoclonal antibody (McAb) 4G10 bound strongly and discretely to the EqSS of permeabilized boar, ram, and bull spermatozoa. It also bound to a small patch on the posterior acrosomal region of permeabilized mouse and rat spermatozoa, suggesting that the EqSS is not restricted to the order Artiodactyla. An anti-HSPA1A (formerly Hsp70) antibody recognized the EqSS in boar spermatozoa. Immunogold labeling with McAb 4G10 localized the tyrosine phosphorylated proteins to the outer acrosomal membrane. This was verified by freeze-fracture electron microscopy, which identified the EqSS in three overlying membranes, the plasma membrane, outer acrosomal membrane, and inner acrosomal membrane. In all five species, tyrosine phosphorylated proteins became restricted to the EqSS during sperm maturation in the epididymis. The major tyrosine phosphorylated proteins in the EqSS of boar and ram spermatozoa were identified by mass spectrometry as orthologs of human SPACA1 (formerly SAMP32). Immunofluorescence with a specific polyclonal antibody localized SPACA1 to the equatorial segment in boar spermatozoa. We speculate that the EqSS is an organizing center for assembly of multimolecular complexes that initiate fusion competence in this area of the plasma membrane following the acrosome reaction.

Long-term culture of fully differentiated adult insect neurons

An identified group of cells, dorsal unpaired median (DUM) neurons, isolated from the central nervous system of adult cockroaches can be grown in vitro for extended periods. These cells often develop morphological characteristics that differ from their in situ appearance but physiological experiments demonstrate that they retain their distinctive in vivo membrane properties. A method for culturing insect CNS neurons, which includes the use of a haemolymph-derived growth-enhancing factor is described, and this technique is compared with other attempts to develop an efficient system for producing in vitro preparations of well-defined, identifiable neurons that would provide a model system for aspects of neuron developmental growth, repair and function.

Glial Cell Involvement in Brain Repair and the Effects of Aging

The integrity of the central nervous system (CNS) is essential for the normal functioning of all aspects of animal behavior from the coordination of segmental muscle contractions in annelid locomotion to the development and maintenance of synaptic pathways involved in human memory and thought. The effects of brain injury are likely, therefore, to be devastating. It follows that repair of damage caused by disease or accident is of enormous importance to the well-being of the individual affected and yet the adult mammalian brain has poor powers of recovery from injury. If nerve tracts in the peripheral nervous system are damaged, it does not follow that the region of injury will not recover its full range of movement since axons in the periphery are able to regrow and reestablish innervation. A similar injury within the CNS, however, will not be repaired in a way that will lead to functional recovery despite the fact that CNS axons are intrinsically capable of growth and that sprouting and extension of neuntes are initiated by injury. The main factor that controls the different outcomes in these two cases is the glial environment, which is permissive of axonal growth in the peripheral nervous system but checks growth within the CNS (1).

Cells at the Boundary

Animals are faced with a barrage of information from their surroundings that they must receive, interpret, and act on to survive. Evolutionary pressures for efficient sensory perception, signal transmission, and information processing giving rapid and appropriate responses to stimuli have resulted in the development of a range of nervous systems across the animal kingdom from the simple nerve nets of coelenterates to the highly complex and centralized systems found in vertebrates and culminating in the daunting level of organization found in the human brain. Such organization requires the formation of extremely precise cellular interactions during embryogenesis and early development when the interrelated growth of neurons and glial cells is manipulated by a whole series of controlling factors expressed at different stages of growth. One of the prices to be paid for such precision may be that, once the central nervous system (CNS) has been laid down, the mechanisms designed to protect its structural integrity are the very factors that prevent regeneration when disease or injury damage neural components. Thus, the incidence of neural regeneration is greater in those phyla whose members have simple nervous systems retaining a high degree of plasticity into maturity. In phyla with highly centralized specialization of the nervous tissue, regeneration, especially in adult animals, may be limited: In mammals, for example, regenerative processes are poorly developed, slow, and very largely confined to the peripheral nervous system (PNS).