Ivan de Curtis

p95-APP1 links membrane transport to Rac-mediated reorganization of actin

Motility requires protrusive activity at the cellular edge, where Rho family members regulate actin dynamics. Here we show that p95-APP1 (ArfGAP-putative, Pix-interacting, paxillin-interacting protein 1), a member of the GIT1/PKL family, is part of a complex that interacts with Rac. Wild-type and truncated p95-APP1 induce actin-rich protrusions mediated by Rac and ADP-ribosylation factor 6 (Arf6). Distinct p95-APP1-derived polypeptides have different distributions, indicating that p95-APP1 cycles between the cell surface and endosomes. Our results show that p95-APP1 functionally interacts with Rac and localizes to endosomal compartments, thus identifying p95-APP1 as a molecular link between actin organization, adhesion, and membrane transport during cell motility.

Generation and Characterization of Rac3 Knockout Mice

ABSTRACT Rac proteins are members of the Rho family of GTPases involved in the regulation of actin dynamics. The three highly homologous Rac proteins in mammals are the ubiquitous Rac1, the hematopoiesis-specific Rac2, and the least-characterized Rac3. We show here that Rac3 mRNA is widely and specifically expressed in the developing nervous system, with highest concentration at embryonic day 13 in the dorsal root ganglia and ventral spinal cord. At postnatal day 7 Rac3 appears particularly abundant in populations of projection neurons in several regions of the brain, including the fifth layer of the cortex and the CA1-CA3 region of the hippocampus. We generated mice deleted for the Rac3 gene with the aim of analyzing the function of this GTPase in vivo. Rac3 knockout animals survive embryogenesis and show no obvious developmental defects. Interestingly, specific behavioral differences were detected in the Rac3-deficient animals, since motor coordination and motor learning on the rotarod was superior to that of their wild-type littermates. No obvious histological or immunohistological differences were observed at major sites of Rac3 expression. Our results indicate that, in vivo, Rac3 activity is not strictly required for normal development in utero but may be relevant to later events in the development of a functional nervous system.

Cell migration: GAPs between membrane traffic and the cytoskeleton

During cell migration, coordination between membrane traffic, cell substrate adhesion and actin reorganization is required for protrusive activity to occur at the leading edge. Actin organization is regulated by Rho family GTPases and, with a contribution from the endocytic cycle, serves to extend the cell front. The details of the molecular mechanisms that direct membrane traffic at sites of adhesion and rearrange actin at the cell edge are still unknown. However, recent findings show that a number of multi‐domain proteins characterized by an ArfGAP domain interact with both actin‐regulating and integrin‐binding proteins, as well as affecting Rac‐mediated protrusive activity and cell migration. Some of these proteins have been shown to localize to endocytic compartments and to have a role in regulating endocytosis. Given the participation of Arf proteins in regulating membrane traffic, one appealing hypothesis is that the ArfGAPs act as molecular devices that coordinate membrane traffic and cytoskeletal reorganization during cell motility.

Functions of Rac GTPases during Neuronal Development

The small GTPases of the Rho family are important regulators of the actin cytoskeleton and are critical for several aspects of neuronal development including the establishment of neuronal polarity, extension of axon and dendrites, neurite branching, axonal navigation and synapse formation. The aim of this review is to present evidence supporting the function of Rac and Rac-related proteins in different aspects of neuronal maturation, based on work performed with organisms including nematodes, Drosophila, Xenopus and mice, and with primary cultures of developing neurons. Three of the 4 vertebrate Rac-related genes, namely Rac1, Rac3 and RhoG, are expressed in the nervous system, and several data support an essential role of all 3 GTPases in distinct aspects of neuronal development and function. Two important points emerge from the analysis presented: highly homologous Rac-related proteins may perform different functions in the developing nervous system; on the other hand, the data also indicate that similar GTPases may perform redundant functions in vivo.The small GTPases of the Rho family are important regulators of the actin cytoskeleton and are critical for several aspects of neuronal development including the establishment of neuronal polarity, ex

Estradiol Induces Differential Neuronal Phenotypes by Activating Estrogen Receptor α or β1

Estrogens are female sex steroids that have a plethora of effects on a wide range of tissues. These effects are mediated through two well characterized intracellular receptors: estrogen receptor α and β (ERα and ERβ, respectively). Because of their high structural homology, it has been argued whether these two receptors may elicit differential biochemical events in estrogen target cells. Here we examine the effect of 17β-estradiol-dependent activation of ERα and ERβ on neurite sprouting, a well known consequence of this sex hormone action in neural cells. In SK-N-BE neuroblastoma cells transfected with ERα or ERβ, 17β-estradiol induces two distinct morphological phenotypes. ERα activation results in increased length and number of neurites, whereas ERβ activation modulates only neurite elongation. By the use of chimeric receptors we demonstrate that the presence of both transcription activation functions located in the NH2-terminus and COOH-terminus of the two ER proteins are necessary for maintaining the ...

Differential distribution of Rac1 and Rac3 GTPases in the developing mouse brain: implications for a role of Rac3 in Purkinje cell differentiation.

Rac3 is one of the three known Rac GTPases in vertebrates. Rac3 shows high sequence homology to Rac1, and its transcript is specifically expressed in the developing nervous system, where its localization and function are unknown. By using Rac3‐specific antibodies, we show that the endogenous Rac3 protein is differentially expressed during mouse brain development, with a peak of expression at times of neuronal maturation and synaptogenesis. Comparison with Rac1 shows clear‐cut differences in the overall distribution of the two GTPases in the developing brain, and in their subcellular distribution in regions of the brain where both proteins are expressed. At P7, Rac3 staining is particularly marked in the deep cerebellar nuclei and in the pons, where it shows a discontinuous distribution around the neuronal cell bodies, in contrast with the diffuse staining of Rac1. Rac3 does not evidently co‐localize with pre‐ and post‐synaptic markers, nor with GFAP‐positive astrocytes, but it clearly co‐localizes with actin filaments, and with the terminal portions of calbindin‐positive Purkinje cell axons in the deep cerebellar nuclei. Our data implicate Rac3 in neuronal differentiation, and support a specific role of this GTPase in actin‐mediated remodelling of Purkinje cell neuritic terminals at time of synaptogenesis.

Cell surface dynamics – how Rho GTPases orchestrate the interplay between the plasma membrane and the cortical cytoskeleton

Summary Small GTPases are known to regulate hundreds of cell functions. In particular, Rho family GTPases are master regulators of the cytoskeleton. By regulating actin nucleation complexes, Rho GTPases control changes in cell shape, including the extension and/or retraction of surface protrusions and invaginations. Protrusion and invagination of the plasma membrane also involves the interaction between the plasma membrane and the cortical cytoskeleton. This interplay between membranes and the cytoskeleton can lead to an increase or decrease in the plasma membrane surface area and its tension as a result of the fusion (exocytosis) or internalization (endocytosis) of membranous compartments, respectively. For a long time, the cytoskeleton and plasma membrane dynamics were investigated separately. However, studies from many laboratories have now revealed that Rho GTPases, their modulation of the cytoskeleton, and membrane traffic are closely connected during the dynamic remodeling of the cell surface. Arf- and Rab-dependent exocytosis of specific vesicles contributes to the targeting of Rho GTPases and their regulatory factors to discrete sites of the plasma membrane. Rho GTPases regulate the tethering of exocytic vesicles and modulate their subsequent fusion. They also have crucial roles in the different forms of endocytosis, where they participate in the sorting of membrane domains as well as the sculpting and sealing of membrane flasks and cups. Here, we discuss how cell surface dynamics depend on the orchestration of the cytoskeleton and the plasma membrane by Rho GTPases.

Essential role of Rac1 and Rac3 GTPases in neuronal development

Rac GTPases are members of the Rho family regulating the actin cytoskeleton and implicated in neuronal development. Ubiquitous Rac1 and neuron‐specific Rac3 GTPases are coexpressed in the developing mammalian brain. We used Cre‐mediated conditional deletion of Rac1 in neurons combined with knockout of neuron‐specific Rac3 to study the role of these GTPases in neural development. We found that lack of both genes causes motor behavioral defects, epilepsy, and premature death of mice. Deletion of either GTPase does not produce evident phenotypes. Double‐knockout mice show specific defects in the development of the hippocampus. Selective impairment of the dorsal hilus of double‐knockout animals is associated with alteration in the formation of the hippocampal circuitry. Axonal pathways to and from the dorsal hilus are affected because of the deficit of hilar mossy cells. Moreover, analysis of Rac function in hippocampal cultures shows that spine formation is strongly hampered only in neurons lacking both Rac proteins. These findings show for the first time that both Rac1 and Rac3 are important for the development of the nervous system, wherein they play complementary roles during late stages of neuronal and brain development.— Corbetta, S.,Gualdoni, S., Ciceri, G., Monari, M., Zuccaro, E., Tybulewicz, V. L. J., De Curtis, I. Essential role of Rac1 and Rac3 GTPases in neuronal development. FASEBJ. 23, 1347–1357 (2009)

Isolation of exocytic carrier vesicles from BHK cells

Newly synthesized cell surface glycoproteins are transported from the trans-Golgi network (TGN) to the plasma membrane in vesicular carriers. Here we describe a cell-free system in which the formation of these carrier vesicles is reconstituted. Vesicle formation and release occurred specifically from the TGN and were dependent on ATP and cytosol. The released vesicles were isolated by density gradient sedimentation and specific immunoadsorption. Electron microscopy demonstrated that the vesicles had a diameter of 84 +/- 6 nm. The immunoisolated vesicles had a highly simplified protein pattern on two-dimensional gel electrophoresis.

Leucine-zipper-mediated homo- and hetero-dimerization of GIT family p95-ARF GTPase-activating protein, PIX-, paxillin-interacting proteins 1 and 2.

ADP-ribosylation factor GTPase-activating proteins (ARFGAPs) of the G-protein-coupled receptor kinase interactor 1/p95 paxillin kinase linker/p95-ARFGAP Pak-interacting exchange factor paxillin-binding protein (APP)-1 family are multidomain proteins, which interact functionally with both ARF and Rac GTPases. These proteins are involved in the dynamic reorganization of adhesion and the cytoskeleton during cell motility. Our previous work [Di Cesare, Paris, Albertinazzi, Dariozzi, Andersen, Mann, Longhi and de Curtis (2000) Nat. Cell Biol. 2, 521-530] has pointed out a role for p95-APP1 in the regulation of ARF6-mediated membrane recycling. These proteins include different domains, and are capable of interacting stably with proteins that are supposed to play a role in the regulation of actin dynamics and adhesion. They contain a coiled-coil region comprising a putative leucine zipper, predicted to be involved in dimerization. In the present study, we have investigated the possibility that these proteins form dimers. Our results show that p95-APP1 forms homodimers and may also form heterodimers with the other member of the family, p95 paxillin kinase linker/p95-APP2. Both homo- and heterodimerization are disrupted by mutation of two leucine residues in the coiled-coil region of p95-APP1. The N-terminal portion of p95-APP1, including the ARFGAP domain, three ankyrin repeats and the Pak-interacting exchange factor-binding region, are not required for dimerization. Evidence is presented for the existence of endogenous oligomeric complexes. The implication of dimerization/oligomerization in the functioning of these proteins is discussed.