Faculty Bibliography

Three tandemly arrayed protocadherin gene clusters (Pcdh-alpha, -beta, -gamma) comprising more than 50 genes are found in human and mouse. Here, we have investigated the expression and distribution of individual gamma-protocadherins (Pcdhs-gamma) in the developing mouse brain. We find that transfection of Pcdh-gamma genes promotes calcium-dependent cell adhesion in HEK 293 cells. Furthermore, Pcdh-gamma can be recruited to synapses of transfected primary hippocampal neurons. Several individual members of the in total 22 Pcdhs-gamma were chosen to examine the expression of the three subfamilies, Pcdh-gammaA, -gammaB, and -gammaC. These Pcdh-gamma transcripts are expressed all over the brain, with minor regional and cell-type specific differences. Interestingly, a distinct, later onset of expression is observed for Pcdh-gammaC5, a gene located at the end of the Pcdh-gamma cluster. Largely overlapping expression patterns of individual Pcdh-gamma proteins are detected with anti-peptide antibodies. Small differences are observed in the staining of dendritic processes and synapse-rich layers. Our results support the idea that Pcdhs-gamma participate in neuronal differentiation and may be implicated in the fine-tuning of neuronal morphology and synaptogenesis. Cell autonomous regulation of transcription might generate the widespread distribution of individual Pcdhs-gamma in the brain, which is strikingly different from the restricted expression patterns observed for classical cadherins. Thus, a defined set of Pcdhs-gamma may engage in neuronal adhesion and signaling on the cellular level.

Latent transforming growth factor (TGF)-beta binding proteins (LTBPs) modulate the secretion and activation of latent TGF-beta. To explore LTBP function in vivo, we created an Ltbp-3(-/-) mouse that has developmental emphysema with decreased septation in terminal alveoli. Differences in distal airspace enlargement were obvious at day 6 after birth. Secondary septation was inhibited, so by days 21 to 28 the mean linear intercept was approximately twofold greater in mutant versus control lungs. There were no differences in lung collagen and elastin, visualized by immunohistochemistry, or in myofibroblast numbers, determined by alpha-smooth muscle actin-positive cells, between mutant or wild-type lungs as the animals aged, other than differences associated with altered lung structure in mutant animals. However, from day 10 there was twice the number of alveolar type II cells in mutant alveoli compared to controls. At days 6 and 10, a transient enhancement in cell proliferation in the mutant lungs was observed by both 5-bromo-2'-deoxy-uridine and proliferating cell nuclear antigen labeling, accompanied by enhanced numbers of terminal dUTP nick-end labeling-positive cells at days 4, 6, and 10. Finally, there was a transient decrease in TGF-beta signaling at days 4 to 6 in Ltbp-3(-/-) lungs. These results indicate that in the absence of Ltbp-3, a temporary decrease in TGF-beta signaling in the lungs at days 4 to 6 alters cell proliferation, correlating with inhibition of septation and developmental emphysema

Relatively little is known about the relationship between cellular lipid composition and the ability of neuroblasts to elaborate axonal and dendritic processes. We have studied the role of cholesterol and non-sterol isoprenoids during neurite outgrowth in PC-12 cells using inhibitors of cholesterol biosynthesis that act at different points in the biosynthetic pathway. We provide evidence that inhibition of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase leads to extensive, sterol-dependent neurite outgrowth, via a mechanism that is independent of the requirement for sterols during proliferation. This effect is prevented by non-sterol mevalonate derivatives, suggesting the involvement of protein prenylation in the regulation of neurite outgrowth. Furthermore, we show that lovastatin inhibits both RhoA activation and Cofilin phosphorylation, while geranylgeraniol reverses these effects. Finally, the effect of geranylgeraniol on neurite outgrowth is prevented by Y-27632, an inhibitor of RhoA kinase. Taken together, our results suggest that inhibition of geranylgeraniol synthesis causes sterol-dependent neurite outgrowth in a process that is mediated by inhibition of RhoA signaling

Macrophage internalization of modified lipoproteins is thought to play a critical role in the initiation of atherogenesis. Two scavenger receptors, scavenger receptor A (SR-A) and CD36, have been centrally implicated in this lipid uptake process. Previous studies showed that these receptors mediated the majority of cholesterol ester accumulation in macrophages exposed to oxidized LDL and that mice with deletions of either receptor exhibited marked reductions in atherosclerosis. This work has contributed to an atherosclerosis paradigm: scavenger receptor-mediated oxidized lipoprotein uptake is required for foam cell formation and atherogenesis. In this study, Apoe-/- mice lacking SR-A or CD36, backcrossed into the C57BL/6 strain for 7 generations, were fed an atherogenic diet for 8 weeks. Hyperlipidemic Cd36-/-Apoe-/- and Msr1-/-Apoe-/- mice showed significant reductions in peritoneal macrophage lipid accumulation in vivo; however, in contrast with previous reports, this was associated with increased aortic sinus lesion areas. Characterization of aortic sinus lesions by electron microscopy and immunohistochemistry showed abundant macrophage foam cells, indicating that lipid uptake by intimal macrophages occurs in the absence of CD36 or SR-A. These data show that alternative lipid uptake mechanisms may contribute to macrophage cholesterol ester accumulation in vivo and suggest that the roles of SR-A and CD36 as proatherosclerotic mediators of modified LDL uptake in vivo need to be reassessed

The role of hemodynamic forces and other signals from circulating blood in guiding the development of the retinal vasculature was examined by following the growth of these vessels in organ cultures. Retinal vascular development in organ cultures was monitored by immunofluorescent staining of retinal whole-mounts using antibodies against ICAM-2, a specific marker for endothelial cells and by vascular adenosine disphosphatase activity. Under culture conditions, the retinal vasculature from mice at postnatal day 3 (P3) grew from the optic nerve area to the edge of the retina in a manner similar to that observed in vivo. Both inner and outer vascular plexuses formed in retinal explants. Within the first few days of organ culture, the initial uniform meshwork of blood vessels was reorganized into arterioles, venules, and capillaries. As in animals, the initial retinal vascular plexus contained abundant vessels, and afterward some vessels regressed leading to the formation of a mature vascular bed. Changes in vascular density due to blood vessel growth and remodeling were confirmed by RT-PCR and Western blot analyses of ICAM-2 mRNA and protein levels, respectively. In addition, during in vitro retinal vascularization, arterioles acquired mural cell coverage, as shown by positive staining for alpha-smooth muscle actin. Thus, blood flow and blood-derived signals were not required for the development and maturation of retinal vessels. In contrast, stability of blood vessels in retinal explants was tightly regulated by endogenous levels of vascular endothelial growth factor-A (VEGF-A). VEGF-A was expressed in the explants throughout the culture period, and addition of neutralizing antibodies against VEGF-A to the organ culture caused a severe regression of blood vessels from the vascular front toward the optic nerve. In contrast, addition of anti-FGF-2 antibodies had no effect on the developing vasculature. Thus, retinal vascular development is dependent on local VEGF-A signals rather than systemic signals

Ras proteins associate with cellular membranes by virtue of a series of post-translational modifications of their C-terminal CAAX sequences. The discovery that two of the three enzymes that modify CAAX proteins are restricted to the endoplasmic reticulum led to the recognition that all nascent Ras proteins transit endomembranes en route to the PM (plasma membrane) and that at steady-state N-Ras and H-Ras are highly expressed on the Golgi apparatus. To test the hypothesis that Ras proteins on internal membranes can signal, we developed a fluorescent probe that reports when and where in living cells Ras becomes active. We found that growth factors stimulated rapid and transient activation of Ras on the PM followed by delayed and sustained activation on the Golgi. We mapped one pathway responsible for this activity as involving PLCgamma (phospholipase Cgamma)/DAG (diacylglycerol)+Ca2+/RasGRP1. Using mammalian cells and fission yeast, we have shown that differential localization of activated Ras preferentially activates distinct signalling pathways. In very recent work, we have found that (i) the subcellular localization of K-Ras can be acutely modulated by phosphorylation of its C-terminal hypervariable region by PKC, (ii) among the membranes upon which phosphorylated K-Ras accumulates is the outer mitochondrial membrane and (iii) phosphorylated, internalized K-Ras promotes apoptosis. Thus the signalling output of Ras depends on its subcellular localization

Extended abstract of a paper presented at Microscopy and Microanalysis 2005 in Honolulu, Hawaii, USA, July 31--August 4, 2005.

For sperm to fertilize eggs, they must first bind to the thick zona pellucida (ZP) that surrounds the plasma membrane of all unfertilized mammalian eggs. An extensive literature suggests that mouse sperm recognize and bind to a specific ZP glycoprotein called mZP3. However, the role of individual ZP glycoproteins in binding of mouse sperm to eggs has been called into question by recent transgenic experiments with null mice. Results of such experiments have been interpreted to mean that binding of sperm depends on the supramolecular structure of the ZP, not on an individual ZP glycoprotein. Here, it is argued that results of these transgenic experiments actually are consistent with the prevailing view of gamete recognition that implicates a specific ZP glycoprotein in both binding of mouse sperm to eggs and induction of the acrosome reaction.

The cell biologist's insight into endosomal diversity, in terms of both form and function, has increased dramatically in the past few years. This understanding has been promoted by the availability of powerful new techniques that allow imaging of both cargo and machinery in the endocytic process in real time, and by our ability to inhibit components of this machinery by RNA interference. The emerging picture from these studies is of a highly complex, dynamic and adaptable endosomal system that interacts at various points with the secretory system of the cell.