Synaptic vesicle tethering and the CaV2.2 distal C-terminal.

Evidence that synaptic vesicles (SVs) can be gated by a single voltage sensitive calcium channel (CaV2.2) predict a molecular linking mechanism or "tether" (Stanley, 1993). Recent studies have proposed that the SV binds to the distal C-terminal on the CaV2.2 calcium channel (Kaeser et al., 2011; Wong et al., 2013) while genetic analysis proposed a double tether mechanism via RIM: directly to the C terminus PDZ ligand domain or indirectly via a more proximal proline rich site (Kaeser et al., 2011). Using a novel in vitro SV pull down binding assay, we reported that SVs bind to a fusion protein comprising the C-terminal distal third (C3, aa 2137-2357; Wong et al., 2013). Here we limit the binding site further to the last 58 aa, beyond the proline rich site, by the absence of SV capture by a truncated C3 fusion protein (aa 2137-2299). To test PDZ-dependent binding we generated two C terminus-mutant C3 fusion proteins and a mimetic blocking peptide (H-WC, aa 2349-2357) and validated these by elimination of MINT-1 or RIM binding. Persistence of SV capture with all three fusion proteins or with the full length C3 protein but in the presence of blocking peptide, demonstrated that SVs can bind to the distal C-terminal via a PDZ-independent mechanism. These results were supported in situ by normal SV turnover in H-WC-loaded synaptosomes, as assayed by a novel peptide cryoloading method. Thus, SVs tether to the CaV2.2 C-terminal within a 49 aa region immediately prior to the terminus PDZ ligand domain. Long tethers that could reflect extended C termini were imaged by electron microscopy of synaptosome ghosts. To fully account for SV tethering we propose a model where SVs are initially captured, or "grabbed," from the cytoplasm by a binding site on the distal region of the channel C-terminal and are then retracted to be "locked" close to the channel by a second attachment mechanism in preparation for single channel domain gating.

Synaptic vesicle capture by CaV2.2 calcium channels.

The fusion of synaptic vesicles (SVs) at the presynaptic transmitter release face is gated by Ca(2) (+) influx from nearby voltage-gated calcium channels (CaVs). Functional studies favor a direct molecular "tethering" attachment and recent studies have proposed a direct link to the channel C-terminal. To test for direct CaV-SV attachment we developed an in vitro assay, termed SV pull-down (SV-PD), to test for capture of purified, intact SVs. Antibody-immobilized presynaptic or expressed CaV2.2 channels but not plain beads, IgG or pre-blocked antibody successfully captured SVs, as assessed byWestern blot for a variety of protein markers. SV-PD was also observed with terminal fusion proteins of the distal half of the C-terminal, supporting involvement of this CaV region in tethering. Thus our results support a model in which the SV tethers directly to the CaV. Since the tip of the C-terminal could extend as far as 200 nm into the cytoplasm, we hypothesize that this link may serve as the initial SV capture mechanism by the release site. Further studies will be necessary to evaluate the molecular basis of C-terminal tethering and whether the SV binds to the channel by additional, shorter-range attachments.

Slow chemical transmission between dorsal root ganglion neuron somata.

Somatic sensory neuron somata are located within the dorsal root ganglia (DRG) and are mostly ensheathed by individual satellite glial cell sheets. It has been noted, however, that a subpopulation of these DRG somata are intimately associated, separated only by a single thin satellite glial cell membrane septum. We set out to test whether such neuron-glial cell-neuron trimers (NGlNs) are also linked functionally. The presence of NGlNs in chick DRGs was confirmed by electron microscopy. Selective satellite glial cell immunostains were identified and were used to image the inter-neuron septa in DRG frozen sections. We used a gentle, dispase-based enzymatic method to isolate chick and rat NGlNs in vitro for double patch clamp recordings. In the majority of pairs tested, an action potential-like stimulus train delivered to one soma resulted in a delayed, noisy and long-duration response in its idle partner. The response to a second stimulus train given minutes later was markedly facilitated. Both bidirectional and unidirectional transmission was observed between the paired neurons. Transmission was chemical and block by the general purinergic blocker suramin implicated ATP as a neurotransmitter. We conclude that the two neuronal somata in the NGlN can communicate by chemical transmission, which may involve a transglial, bi-synaptic pathway. This novel soma-to-soma transmission reflects a novel form of processing that may play a role in sensory disorders in the DRG and interneuron communication in the central nervous system.

Ancient origins determine global biogeography of hot and cold desert cyanobacteria.

Factors governing large-scale spatio-temporal distribution of microorganisms remain unresolved, yet are pivotal to understanding ecosystem value and function. Molecular genetic analyses have focused on the influence of niche and neutral processes in determining spatial patterns without considering the temporal scale. Here, we use temporal phylogenetic analysis calibrated using microfossil data for a globally sampled desert cyanobacterium, Chroococcidiopsis, to investigate spatio-temporal patterns in microbial biogeography and evolution. Multilocus phylogenetic associations were dependent on contemporary climate with no evidence for distance-related patterns. Massively parallel pyrosequencing of environmental samples confirmed that Chroococcidiopsis variants were specific to either hot or cold deserts. Temporally scaled phylogenetic analyses showed no evidence of recent inter-regional gene flow, indicating populations have not shared common ancestry since before the formation of modern continents. These results indicate that global distribution of desert cyanobacteria has not resulted from widespread contemporary dispersal but is an ancient evolutionary legacy. This highlights the importance of considering temporal scales in microbial biogeography.

N-type Ca2+ channels carry the largest current: implications for nanodomains and transmitter release.

Presynaptic terminals favor intermediate-conductance Ca(V)2.2 (N type) over high-conductance Ca(V)1 (L type) channels for single-channel, Ca(2+) nanodomain-triggered synaptic vesicle fusion. However, the standard Ca(V)1>Ca(V)2>Ca(V)3 conductance hierarchy is based on recordings using nonphysiological divalent ion concentrations. We found that, with physiological Ca(2+) gradients, the hierarchy was Ca(V)2.2>Ca(V)1>Ca(V)3. Mathematical modeling predicts that the Ca(V)2.2 Ca(2+) nanodomain, which is ∼25% more extensive than that generated by Ca(V)1, can activate a calcium-fusion sensor located on the proximal face of the synaptic vesicle.

Hypolithic microbial community of quartz pavement in the high-altitude tundra of central Tibet.

The hypolithic microbial community associated with quartz pavement at a high-altitude tundra location in central Tibet is described. A small-scale ecological survey indicated that 36% of quartz rocks were colonized. Community profiling using terminal restriction fragment length polymorphism revealed no significant difference in community structure among a number of colonized rocks. Real-time quantitative PCR and phylogenetic analysis of environmental phylotypes obtained from clone libraries were used to elucidate community structure across all domains. The hypolithon was dominated by cyanobacterial phylotypes (73%) with relatively low frequencies of other bacterial phylotypes, largely represented by the chloroflexi, actinobacteria, and bacteriodetes. Unidentified crenarchaeal phylotypes accounted for 4% of recoverable phylotypes, while algae, fungi, and mosses were indicated by a small fraction of recoverable phylotypes.

Comparison of the DiagCor GenoFlow Human Papillomavirus Array Test and Roche Linear Array HPV Genotyping Test.

Persistent infection of high-risk (HR) human papillomavirus (HPV) infection has been widely associated with cervical cancer. Monitoring HPV infection is therefore an important step against cervical cancer development. The DiagCor GenoFlow Human Papillomavirus Array Test (GenoFlow) is a novel HPV test based on PCR and "Flow-through" hybridization that can identify 33 HPV subtypes in 3 hours. In the present study, the GenoFlow Test was evaluated by comparing the genotyping results of 100 samples with Roche Linear Array HPV Genotyping Test (LA). The tests showed good agreement in detection of HPV-positive samples (concordance rate=95%, Cohen's Kappa=0.896), with good agreement in detection of HR HPVs (Cohen's Kappa=0.876). The GenoFlow Test showed high sensitivity (95%), high specificity (95%), low false positive rate (3.33%) and low false negative rate (7.50%). In conclusion, the novel GenoFlow Test showed comparable clinical performance to LA test, and offers advantages of reduction in turnaround time and manpower.

Endolithic microbial colonization of limestone in a high-altitude arid environment.

The morphology of endolithic colonization in a limestone escarpment and surrounding rocky debris (termed float) at a high-altitude arid site in central Tibet was documented using scanning electron microscopy. Putative lichenized structures and extensive coccoid bacterial colonization were observed. Absolute and relative abundance of rRNA gene signatures using real-time quantitative polymerase chain reaction and phylogenetic analysis of environmental phylotypes were used to characterize community structure across all domains. Escarpment endoliths were dominated by eukaryotic phylotypes suggestive of lichenised associations (a Trebouxia lichen phycobiont and Leptodontidium lichen mycobiont), whereas float endoliths were dominated by bacterial phylotypes, including the cyanobacterium Chroococcidiopsis plus several unidentified beta proteobacteria and crenarchaea. Among a range of abiotic variables tested, ultraviolet (UV) transmittance by rock substrates was the factor best able to explain differences in community structure, with eukaryotic lichen phylotypes more abundant under conditions of greater UV-exposure compared to prokaryotes. Variously pigmented float rocks did not support significantly different communities. Estimates of in situ carbon fixation based upon (14)C radio-labelled bicarbonate uptake indicated endolithic productivity of approximately 2.01 g C/m(2)/year(-1), intermediate between estimates for Antarctic and temperate communities.

Rab3a interacting molecule (RIM) and the tethering of pre-synaptic transmitter release site-associated CaV2.2 calcium channels.

Biochemical and physiological evidence suggest that pre-synaptic calcium channels are attached to the transmitter release site within the active zone by a molecular tether. A recent study has proposed that 'Rab3a Interacting Molecule' (RIM) serves as the tether for CaV2.1 channels in mouse brain, based in part on biochemical co-immunoprecipitation (co-IP) using a monoclonal antibody, mRIM. We previously argued against this idea for CaV2.2 calcium channel at chick synapses based on experiments using a different anti-RIM antibody, pRIM1,2: while staining for the two proteins co-localized and co-varied at the transmitter release face, consistent with an association, they failed to co-IP from a synaptosome membrane lysate. RIM is, however, a family of proteins and we tested the possibility that the mRIM antibody used in the more recent study identifies a particular channel-tethering variant. We find that co-immunostaining with mRIM and anti-CaV2.2 antibody neither co-localized nor co-varied at the transmitter release face and the two proteins did not co-IP, arguing against a common protein complex and a key CaV2.2 scaffolding role for RIM at the active zone. The differing results might be reconciled, however, in a model where a RIM family member contributes to a protein bridge that anchors the pre-fusion secretory vesicle to the calcium channel protein complex.