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 Ca(2+)-dependent phosphatase calcineurin (Silverman-Gavrila and Charlton, 2009). Since the fast Ca(2+) chelator BAPTA-AM inhibits LFD but the slow chelator EGTA-AM does not, the Ca(2+) sensor for LFD may be close to a Ca(2+) source at active zones. Calcineurin can be activated by the Ca(2+)-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 Ca(2+) and calpain inhibitors. Western blot analysis of CNS extract showed that proteolysis of calcineurin to a fragment consistent with the constitutively active form required Ca(2+) 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 Ca(2+) signaling activates presynaptic calpain and calcineurin at active zones and causes changes of tubulin cytoskeleton.

Calcineurin and cytoskeleton in low-frequency depression.

Transmitter release at high probability phasic synapses of crayfish neuromuscular junctions depresses by over 50% in 60 min when stimulated at 0.2 Hz. Inhibition of the protein phosphatase calcineurin by intracellular pre-synaptic injection of autoinhibitory peptide inhibited low-frequency depression (LFD) and resulted in facilitation of transmitter release. Since this inhibitor had no major effects when injected into the post-synaptic cell, only pre-synaptic calcineurin activity is necessary for LFD. To examine changes in phosphoproteins during LFD we performed a phosphoproteomic screen on proteins extracted from motor axons and nerve terminals after LFD induction or treatment with various drugs that affect kinase and phosphatase activity. Proteins separated by PAGE were stained with phospho-specific/total protein ratio stains (Pro-Q Diamond/SYPRO Ruby) to identify protein bands for analysis by mass spectrometry. Phosphorylation of actin and tubulin decreased during LFD, but increased when calcineurin was blocked. Tubulin and phosphoactin immunoreactivity in pre-synaptic terminals were also reduced after LFD. The actin depolymerizing drugs cytochalasin and latrunculin and the microtubule stabilizer taxol inhibited LFD. Therefore, dephosphorylation of pre-synaptic actin and tubulin and consequent changes in the cytoskeleton may regulate LFD. LFD is unlike long-term depression found in mammalian synapses because the latter requires in most instances post-synaptic calcineurin activity.Thus, this simpler invertebrate synapse discloses a novel pre-synaptic depression mechanism.

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 approximately 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.

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.

An IP3-activated Ca2+ channel regulates fungal tip growth.

Hyphal extension in fungi requires a tip-high Ca(2+) gradient, which is generated and maintained internally by inositol (1,4,5)-trisphosphate (IP(3))-induced Ca(2+) release from tip-localized vesicles and subapical Ca(2+) sequestration. Using the planar bilayer method we demonstrated the presence of two types of IP(3)-activated Ca(2+) channels in Neurospora crassa membranes with different conductances: one low (13 picosiemens), the other high (77 picosiemens). On sucrose density gradients the low conductance channel co-localized with endoplasmic reticulum and plasma membrane, and the high conductance channel co-localized with vacuolar membranes. We correlated the effect of inhibitors on channel activity with their effect on hyphal growth and Ca(2+) gradients. The inhibitor of IP(3)-induced Ca(2+) release, 2-aminoethoxidiphenylborate (2-APB), inhibits both channels, while heparin, 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate, hydrochloride (TMB-8) and dantrolene inhibit only the large conductance channel. Because 2-APB inhibits hyphal growth and dissipates the tip-high cytosolic [Ca(2+)] gradient, whereas heparin microinjection, TMB-8 and dantrolene treatments do not affect growth, we suggest that the small conductance channel generates the obligatory tip-high Ca(2+) gradient during hyphal growth. Since IP(3) production must be catalyzed by tip-localized phospholipase C, we show that a number of phospholipase C inhibitors [neomycin, 1-[6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]- 1H-pyrrole-2,5-dione (U-73122) (but not the inactive pyrrolidine U-73343), 3-nitrocoumarin] inhibit hyphal growth and affect, similarly to 2-APB, the location of vesicular Ca(2+) imaged by chlortetracycline staining.

Calcium gradient dependence of Neurospora crassa hyphal growth.

A tip-high cytoplasmic calcium gradient has been identified as a requirement for hyphal growth in the fungus Neurospora crassa. The Ca2+ gradient is less steep compared to wall vesicle, wall incorporation and vesicular Ca2+ gradients, but this can be explained by Ca2+ diffusion. Analysis of the relation between the rate of hyphal growth and the spatial distribution of tip-localized calcium indicates that hyphal growth rates depend upon the tip-localized calcium concentration. It is not the steepness of the calcium gradient, but tip-localized calcium and the difference in tip-localized calcium versus subapical calcium concentration which correlate closely with hyphal growth rate. A minimal concentration difference between the apex and subapical region of 30 nM is required for growth to occur. The calcium concentration dependence of growth may relate directly to biochemical functions of calcium in hyphal extension, such as vesicle fusion and enzyme activation during cellular expansion. Initiation of tip growth may rely upon random Ca2+ motions causing localized regions of elevated calcium. Continued hyphal expansion may activate a stretch-activated phospholipase C which would increase tip-localized inositol 1,4,5-trisphosphate (IP3). Hyphal expansion, induced by mild hypoosmotic treatment, does increase diacylglycerol, the other product of phospholipase C activity. This is consistent with evidence that IP3-activated Ca2+ channels generate and maintain the tip-high calcium gradient.