Lorelei Silverman-Gavrila

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.

Phosphorylation-Dependent Low-Frequency Depression at Phasic Synapses of a Crayfish Motoneuron

Extremes in presynaptic differentiation can be studied at the crayfish leg extensor muscle where, on the same muscle fiber, one motoneuron makes “phasic” depressing synapses that have a high probability of neurotransmitter release and another motoneuron makes “tonic,” low-probability, facilitating synapses. The large motor axons permit intracellular access to presynaptic sites. We examined the role of phosphorylation during low-frequency depression (LFD) in the relatively little studied phasic synapses. LFD occurs with stimulation at 0.2 Hz and develops with time constants of 4 and 105 min to reach >50% depression of transmitter release in 60 min similar to long-term depression in mammals. LFD is not associated with changes in postsynaptic sensitivity to transmitter and thus is a presynaptic event, although it is not accompanied by changes in the presynaptic action potential. Blockade of protein kinases accelerated the slow phase of LFD, but stimulation of kinases reduced depression. Blockade of protein phosphatases 1A/2A reversed the slow phase. When calcineurin was inhibited, both phases of LFD were abolished, and facilitation occurred instead. Immunostaining showed calcineurin-like immunoreactivity in synaptic terminals. Recovery from LFD occurred in ∼1 h if stimulation frequency was reduced to 0.0016 Hz. Recovery was blocked by kinase inhibition. This study shows that phosphorylation-dependent mechanisms are involved in LFD and suggests that exocytosis is controlled by conditions that shift the balance between phosphorylated and unphosphorylated substrates. The shift can occur by alteration in the relative activities of protein kinases and phosphatases.

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.

Calcineurin and cytoskeleton in low-frequency depression.

J. Neurochem. (2009) 109, 716–732.

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.

Calcium, Calpain, and Calcineurin in Low-Frequency Depression of Transmitter Release

Low-frequency depression (LFD) of transmitter release occurs at phasic synapses with stimulation at 0.2 Hz in both isolated crayfish (Procambarus clarkii) neuromuscular junction (NMJ) preparations and in intact animals. LFD is regulated by presynaptic activity of the Ca2+-dependent phosphatase calcineurin (Silverman-Gavrila and Charlton, 2009). Since the fast Ca2+ chelator BAPTA-AM inhibits LFD but the slow chelator EGTA-AM does not, the Ca2+ sensor for LFD may be close to a Ca2+ source at active zones. Calcineurin can be activated by the Ca2+-activated protease calpain, and immunostaining showed that both proteins are present at nerve terminals. Three calpain inhibitors, calpain inhibitor I, MDL-28170, and PD150606, but not the control compound PD145305, inhibit LFD both in the intact animal as shown by electromyograms and by intracellular recordings at neuromuscular junctions. Analysis of mini-EPSPs indicated that these inhibitors had minimal postsynaptic effects. Proteolytic activity in CNS extract, detected by a fluorescent calpain substrate, was modulated by Ca2+ and calpain inhibitors. Western blot analysis of CNS extract showed that proteolysis of calcineurin to a fragment consistent with the constitutively active form required Ca2+ and was blocked by calpain inhibitors. Inhibition of LFD by calpain inhibition blocks the reduction in phosphoactin and the depolymerization of tubulin that normally occurs in LFD, probably by blocking the dephosphorylation of cytoskeletal proteins by calcineurin. In contrast, high-frequency depression does not involve protein phosphorylation- or calpain-dependent mechanisms. LFD may involve a specific pathway in which local Ca2+ signaling activates presynaptic calpain and calcineurin at active zones and causes changes of tubulin cytoskeleton.

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 …