Rosalind Silverman-Gavrila

Anillin-mediated Targeting of Peanut to Pseudocleavage Furrows Is Regulated by the GTPase Ran

During early development in Drosophila, pseudocleavage furrows in the syncytial embryo prevent contact between neighboring spindles, thereby ensuring proper chromosome segregation. Here we demonstrate that the GTPase Ran regulates pseudocleavage furrow organization. Ran can exert control on pseudocleavage furrows independently of its role in regulating the microtubule cytoskeleton. Disruption of the Ran pathway prevented pseudocleavage furrow formation and restricted the depth and duration of furrow ingression of those pseudocleavage furrows that did form. We found that Ran was required for the localization of the septin Peanut to the pseudocleavage furrow, but not anillin or actin. Biochemical assays revealed that the direct binding of the nuclear transport receptors importin alpha and beta to anillin prevented the binding of Peanut to anillin. Furthermore, RanGTP reversed the inhibitory action of importin alpha and beta. On expression of a mutant form of anillin that lacked an importin alpha and beta binding site, inhibition of Ran no longer restricted the depth and duration of furrow ingression in those pseudocleavage furrows that formed. These data suggest that anillin and Peanut are involved in pseudocleavage furrow ingression in syncytial embryos and that this process is regulated by Ran.

Homotypic and Endothelial Cell Adhesions via N-Cadherin Determine Polarity and Regulate Migration of Vascular Smooth Muscle Cells

Migration of smooth muscle cells from the arterial media to the intima is central to several vascular pathologies including restenosis. This study demonstrates that, like directional migration of other cells, smooth muscle migration is accompanied by a dramatic, polarized reorganization of the cell cytoskeleton that is accompanied by activation of the Rho GTPase Cdc42 and inactivation of glycogen synthase kinase-3&bgr;. We also show, for the first time, that signals generated at the posterior-lateral aspects of wound edge cells by the cell-cell adhesion molecule N-cadherin are required for polarization and rapid migration of vascular smooth muscle. Importantly, when a cohort of migrating smooth muscle cells encounter CHO cells or the A10 smooth muscle cell line, neither of which expresses N-cadherin, polarity is only slightly suppressed. However, when smooth muscle cells encounter stably transfected, N-cadherin-expressing A10 cells or (N-cadherin-expressing) vascular endothelium, they rapidly lose their polarized phenotype. The latter finding indicates that endothelial signaling to innermost smooth muscle cells via N-cadherin may be critical to normal vessel wall stability. We infer that asymmetrical distribution of N-cadherin is necessary for the establishment of cell polarity during migration and that N-cadherin ligation is highly effective in abrogating polarized migration. Finally, we showed that endothelial cell polarity does not depend on N-cadherin; therefore, this molecule may be an attractive target for therapies to prevent restenosis without suppressing endothelial repair and risking late thrombosis.

Human-Chromatin-Related Protein Interactions Identify a Demethylase Complex Required for Chromosome Segregation

Chromatin regulation is driven by multicomponent protein complexes, which form functional modules. Deciphering the components of these modules and their interactions is central to understanding the molecular pathways these proteins are regulating, their functions, and their relation to both normal development and disease. We describe the use of affinity purifications of tagged human proteins coupled with mass spectrometry to generate a protein-protein interaction map encompassing known and predicted chromatin-related proteins. On the basis of 1,394 successful purifications of 293 proteins, we report a high-confidence (85% precision) network involving 11,464 protein-protein interactions among 1,738 different human proteins, grouped into 164 often overlapping protein complexes with a particular focus on the family of JmjC-containing lysine demethylases, their partners, and their roles in chromatin remodeling. We show that RCCD1 is a partner of histone H3K36 demethylase KDM8 and demonstrate that both are important for cell-cycle-regulated transcriptional repression in centromeric regions and accurate mitotic division.

Septins: New Microtubule Interacting Partners

Originally characterized as regulators of cytokinesis, septins were later implicated in other cellular processes. Recent studies show that septins have a broader role in microtubule-dependent processes, such as karyokinesis, exocytosis, and maintenance of cell shape. Many members of the septin family have been shown to colocalize or interact with the microtubule cytoskeleton, suggesting that these might be general properties of septins. Septins could play an important role in regulating microtubule dynamics by interacting with microtubule-associated proteins (MAPs) that modulate microtubule stability. Being able to associate with both microtubules and actin, septins can play an important role as adaptors between the two cytoskeletons and as regulators of processes in which both actin and microtubules are involved. As septins are associated with various neurodegenerative diseases and cancer, a better understanding of the biology of septins and their interactions with microtubules is important in order to develop possible therapeutic strategies for these diseases.

Rear polarization of the microtubule-organizing center in neointimal smooth muscle cells depends on PKCα, ARPC5, and RHAMM.

Directed migration of smooth muscle cells (SMCs) from the media to the intima in arteries occurs during atherosclerotic plaque formation and during restenosis after angioplasty or stent application. The polarized orientation of the microtubule-organizing center (MTOC) is a key determinant of this process, and we therefore investigated factors that regulate MTOC polarity in vascular SMCs. SMCs migrating in vivo from the medial to the intimal layer of the rat carotid artery following balloon catheter injury were rear polarized, with the MTOC located posterior of the nucleus. In tissue culture, migrating neointimal cells maintained rear polarization, whereas medial cells were front polarized. Using phosphoproteomic screening and mass spectrometry, we identified ARPC5 and RHAMM as protein kinase C (PKC)-phosphorylated proteins associated with rear polarization of the MTOC in neointimal SMCs. RNA silencing of ARPC5 and RHAMM, PKC inhibition, and transfection with a mutated nonphosphorylatable ARPC5 showed that these proteins regulate rear polarization by organizing the actin and microtubule cytoskeletons in neointimal SMCs. Both ARPC5 and RHAMM, in addition to PKC, were required for migration of neointimal SMCs.

Cadherins at cell-autonomous membrane contacts control macropinocytosis

Cadherins aggregate and stabilize cell–cell junctions through interactions with adjacent cells. In addition, N-cadherin and E-cadherin concentrate at free edges or at the lamellipodia of migrating cells and are found within large vesicles called macropinosomes, which develop from membrane ruffles. The binding properties of cadherins have not previously been associated with the localization of cadherins at membrane ruffles; however, we report that the dorsal, ventral and lateral membrane contacts that occur as a result of the overlap of membrane ruffles aggregate N-cadherin, and that both N-cadherin and E-cadherin promote macropinosome closure and fluid-phase uptake in macropinosomes. These data reveal a previously unsuspected function for cadherin-mediated cell–cell adhesion molecules in the closure of cell-autonomous membrane contacts at membrane ruffles, resulting in macropinocytosis.

Spectrin alpha is important for rear polarization of the microtubule organizing center during migration and spindle pole assembly during division of neointimal smooth muscle cells.

Directed migration of smooth muscle cells (SMCs) from the media to the intima and their subsequent proliferation are key events in atherosclerosis as these cells contribute to the bulk and stability of atheromatous plaques. We showed previously that two cytoskeleton‐associated proteins, RHAMM and ARPC5, play important roles in rear polarization of the microtubule organizing centre (MTOC), directed migration, and in maintaining cell division fidelity. These proteins were analyzed to predict additional potential interacting partners using the bioinformatics programs BLAST, ClustalW, and PPI Spider. We identified spectrin alpha, a protein with a known role in actin polymerization as part of the pathway. We show that in migrating SMCs spectrin alpha localizes at the nodes of the actin net, and it partially colocalizes with RHAMM in the perinuclear region. In dividing SMCs spectrin alpha is present at spindle poles and midbody. Moreover, we show that spectrin alpha and RHAMM interact in a complex. Using siRNA to knockdown spectrin disrupted SMC migration, MTOC polarization, and the assembly of a polygonal actin net dorsolateral of the nucleus. Spectrin alpha knockdown also disrupted the organization of the bipolar spindle, chromosome division, and cytokinesis during cell division. The identification of interacting partners such as spectrin alpha and the decoding of pathways involved in polarity regulation during the migration of smooth muscle cells in atherosclerosis is important for identifying atherosclerosis biomarkers and developing therapeutic agents to block atherosclerotic plaque formation.

Depression in the fly.

Editors Note: These short reviews of a recent paper in the Journal, written exclusively by graduate students or postdoctoral fellows, are intended to mimic the journal clubs that exist in your own departments or institutions. For more information on the format and purpose of the Journal Club, please see A PubMed search for long-term depression (LTD) finds 8703 articles, reflecting the interest this putative mechanism for learning and memory generates among neuroscientists. LTD has been characterized in the mammalian hip-pocampus and cerebellum, and its induction involves regulation of protein phos-phorylation by protein kinases and phosphatases (Mulkey et al., 1994; Linden and Connor, 1995). In arthropods, short-term depression at low-frequency stimulation has been reported at the crayfish (Silverman-Gavrila et al., 2005) and Drosophila neuromuscu-lar junction (NMJ) (Wu et al., 2005). However, Guo and Zhong (2006), in their recent Journal of Neuroscience article, now report LTD properties at Drosophila larval NMJs. Their study provides a direct demonstration that the highly conserved protein kinase B/Akt, known to modulate neurotransmitter receptors (Hou and Klann, 2004), is required for induction of LTD but not short-term plasticity. Dro-sophila NMJs resemble mammalian central glutamatergic synapses in that they share various signaling pathways underlying LTD (Guo and Zhong, 2006). This preparation offers advantages for exploring the molecular mechanism of LTD because presynaptic boutons at identifiable synapses are readily accessible for electro-physiological analysis, unlike most synap-tic boutons in the mammalian brain. Combined with powerful genetic and biochemical manipulation, this may allow a multilevel integrative approach for understanding gene function in synaptic physiology and behavioral plasticity. In their experiments, Guo and Zhong (2006) found that LTD was induced at Drosophila NMJ of muscle M12 by 30 Hz stimulation for 20 s in 0.4 mM external [Ca 2ϩ ] [Guo and Zhong (2006), their Fig. 1 A–C (EJC) amplitude decayed slightly after long recording periods, but to a lesser extent than during LTD induced at high frequency. This is most likely attributable to a reduction in the quantal amplitude Next, the authors showed that stimulation frequency rather than the total number of stimuli was crucial for LTD in-induced at 20 Hz stimulation for 20 s, and it reached a plateau at 30 –50 Hz. In interpreting their results, the authors carefully addressed three concerns: (1) that LTD can be induced at different muscle fibers with different dynamics (LTD and short-term depression at M12 and only LTD at (2) that LTD did …