Alexander Asanov

Visualizing the store-operated channel complex assembly in real time: Identification of SERCA2 as a new member

Depletion of intracellular calcium stores leads to the activation of calcium influx via the so-called store-operated channels (SOCs). Recent evidence positions Orai proteins as the putative channels responsible for this process. The stromal interacting molecule (STIM1) has been recently identified as the calcium sensor located at the endoplasmic reticulum (ER), and responsible for communicating the deplete state of calcium stores to Orai at the plasma membrane (PM). However, recent experimental findings suggest that Orai and STIM1 are only part of a larger molecular complex required to modulate store-operated calcium entry (SOCE). In the present study we describe the assembly of the several of the components from the SOC complex in real-time, utilizing a novel imaging method. Using FRET imaging we show that under resting conditions (with calcium stores replenished) STIM1 travels continuously through the ER associated to the microtubule tracking protein, EB1. Upon depletion of the ER STIM1 dissociates from EB1 and aggregates into macromolecular complexes at the ER which includes the microsomal calcium ATPase. This association follows the assembly of Orai into macromolecular aggregates at the PM. We show that STIM1-Orai association follows a similar time course as that of Orai aggregation at the PM. During this last step of the process, calcium-selective, whole-cell inward currents developed, simultaneously. We show that this process is fully reversible. Replenishing intracellular calcium stores induces STIM1-Orai complex dissociation and shuts down inward currents. Under these conditions STIM1 re-associates to EB1, and reinitiates its travel through the ER.

Combined single channel and single molecule detection identifies subunit composition of STIM1-activated transient receptor potential canonical (TRPC) channels.

Depletion of intracellular calcium ion stores initiates a rapid cascade of events culminating with the activation of the so-called Store-Operated Channels (SOC) at the plasma membrane. Calcium influx via SOC is essential in the initiation of calcium-dependent intracellular signaling and for the refilling of internal calcium stores, ensuring the regeneration of the signaling cascade. In spite of the significance of this evolutionary conserved mechanism, the molecular identity of SOC has been the center of a heated controversy spanning over the last 20 years. Initial studies positioned some members of the transient receptor potential canonical (TRPC) channel superfamily of channels (with the more robust evidence pointing to TRPC1) as a putative SOC. Recent evidence indicates that Stromal Interacting Molecule 1 (STIM1) activates some members from the TRPC family of channels. However, the exact subunit composition of TRPC channels remains undetermined to this date. To identify the subunit composition of STIM1-activated TRPC channels, we developed novel method, which combines single channel electrophysiological measurements based on the patch clamp technique with single molecule fluorescence imaging. We termed this method Single ion Channel Single Molecule Detection technique (SC-SMD). Using SC-SMD method, we have obtained direct evidence of the subunit composition of TRPC channels activated by STIM1. Furthermore, our electrophysiological-imaging SC-SMD method provides evidence at the molecular level of the mechanism by which STIM1 and calmodulin antagonize to modulate TRPC channel activity.

A Cholesterol Recognition Amino Acid Consensus Domain in GP64 Fusion Protein Facilitates Anchoring of Baculovirus to Mammalian Cells

ABSTRACT Baculoviridae is a large family of double-stranded DNA viruses that selectively infect insects. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the best-studied baculovirus from the family. Many studies over the last several years have shown that AcMNPV can enter a wide variety of mammalian cells and deliver genetic material for foreign gene expression. While most animal viruses studied so far have developed sophisticated mechanisms to selectively infect specific cells and tissues in an organism, AcMNPV can penetrate and deliver foreign genes into most cells studied to this date. The details about the mechanisms of internalization have been partially described. In the present study, we have identified a cholesterol recognition amino acid consensus (CRAC) domain present in the AcMNPV envelope fusion protein GP64. We demonstrated the association of a CRAC domain with cholesterol, which is important to facilitate the anchoring of the virus at the mammalian cell membrane. Furthermore, this initial anchoring favors AcMNPV endocytosis via a dynamin- and clathrin-dependent mechanism. Under these conditions, efficient baculovirus-driven gene expression is obtained. In contrast, when cholesterol is reduced from the plasma membrane, AcMNPV enters the cell via a dynamin- and clathrin-independent mechanism. The result of using this alternative internalization pathway is a reduced level of baculovirus-driven gene expression. This study is the first to document the importance of a novel CRAC domain in GP64 and its role in modulating gene delivery in AcMNPV.

A Platform for Combined DNA and Protein Microarrays Based on Total Internal Reflection Fluorescence

We have developed a novel microarray technology based on total internal reflection fluorescence (TIRF) in combination with DNA and protein bioassays immobilized at the TIRF surface. Unlike conventional microarrays that exhibit reduced signal-to-background ratio, require several stages of incubation, rinsing and stringency control, and measure only end-point results, our TIRF microarray technology provides several orders of magnitude better signal-to-background ratio, performs analysis rapidly in one step, and measures the entire course of association and dissociation kinetics between target DNA and protein molecules and the bioassays. In many practical cases detection of only DNA or protein markers alone does not provide the necessary accuracy for diagnosing a disease or detecting a pathogen. Here we describe TIRF microarrays that detect DNA and protein markers simultaneously, which reduces the probabilities of false responses. Supersensitive and multiplexed TIRF DNA and protein microarray technology may provide a platform for accurate diagnosis or enhanced research studies. Our TIRF microarray system can be mounted on upright or inverted microscopes or interfaced directly with CCD cameras equipped with a single objective, facilitating the development of portable devices. As proof-of-concept we applied TIRF microarrays for detecting molecular markers from Bacillus anthracis, the pathogen responsible for anthrax.

A cholesterol-binding domain in STIM1 modulates STIM1-Orai1 physical and functional interactions.

STIM1 and Orai1 are the main components of a widely conserved Calcium influx pathway known as store-operated calcium entry (SOCE). STIM1 is a calcium sensor, which oligomerizes and activates Orai channels when calcium levels drop inside the endoplasmic reticulum (ER). The series of molecular rearrangements that STIM1 undergoes until final activation of Orai1 require the direct exposure of the STIM1 domain known as SOAR (Stim Orai Activating Region). In addition to these complex molecular rearrangements, other constituents like lipids at the plasma membrane, play critical roles orchestrating SOCE. PI(4,5)P2 and enriched cholesterol microdomains have been shown as important signaling platforms that recruit the SOCE machinery in steps previous to Orai1 activation. However, little is known about the molecular role of cholesterol once SOCE is activated. In this study we provide clear evidence that STIM1 has a cholesterol-binding domain located inside the SOAR region and modulates Orai1 channels. We demonstrate a functional association of STIM1 and SOAR to cholesterol, indicating a close proximity of SOAR to the inner layer of the plasma membrane. In contrast, the depletion of cholesterol induces the SOAR detachment from the plasma membrane and enhances its association to Orai1. These results are recapitulated with full length STIM1.

A relay mechanism between EB1 and APC facilitate STIM1 puncta assembly at endoplasmic reticulum-plasma membrane junctions

The assembly of STIM1 protein puncta near endoplasmic reticulum-plasma membrane (ER-PM) junctions is required for optimal activation of store-operated channels (SOC). The mechanisms controlling the translocation of STIM1 puncta to ER-PM junctions remain largely unknown. In the present study, we have explored the role of the microtubule binding protein adenomatous polyposis coli (APC), on STIM1 puncta and store-operated calcium entry (SOCE). APC-depleted cells showed reduced STIM1 puncta near ER-PM junctions, instead puncta is found at the ER surrounding the cell nucleus. Reduced STIM1 puncta near ER-PM junctions in APC-depleted cells correlates with a strong inhibition of SOCE and diminished Orai whole-cell currents. Immunoprecipitation and confocal microscopy co-localization studies indicate that, upon depletion of the ER, STIM1 dissociates from EB1 and associates to APC. Deletion analysis identified an APC-binding domain in the carboxyl terminus of STIM1 (STIM1 650-685). These results together position APC as an important element in facilitating the translocation of STIM1 puncta near ER-PM junctions, which in turn is required for efficient SOCE and Orai activation upon depletion of the ER.

Cholesterol modulates the cellular localization of Orai1 channels and its disposition among membrane domains

Store Operated Calcium Entry (SOCE) is one of the most important mechanisms for calcium mobilization in to the cell. Two main proteins sustain SOCE: STIM1 that acts as the calcium sensor in the endoplasmic reticulum (ER) and Orai1 responsible for calcium influx upon depletion of ER. There are many studies indicating that SOCE is modulated by the cholesterol content of the plasma membrane (PM). However, a myriad of questions remain unanswered concerning the precise molecular mechanism by which cholesterol modulates SOCE. In the present study we found that reducing PM cholesterol results in the internalization of Orai1 channels, which can be prevented by overexpressing caveolin 1 (Cav1). Furthermore, Cav1 and Orai1 associate upon SOCE activation as revealed by FRET and coimmunoprecipitation assays. The effects of reducing cholesterol were not limited to an increased rate of Orai1 internalization, but also, affects the lateral movement of Orai1, inducing movement in a linear pattern (unobstructed diffusion) opposite to basal cholesterol conditions were most of Orai1 channels moves in a confined space, as assessed by Fluorescence Correlation Spectroscopy, Cav1 overexpression inhibited these alterations maintaining Orai1 into a confined and partially confined movement. These results not only highlight the complex effect of cholesterol regulation on SOCE, but also indicate a direct regulatory effect on Orai1 localization and compartmentalization by this lipid.

A novel family of mammalian transmembrane proteins involved in cholesterol transport

Cholesterol is an essential compound in mammalian cells because it is involved in a wide range of functions, including as a key component of membranes, precursor of important molecules such as hormones, bile acids and vitamin D. The cholesterol transport across the circulatory system is a well-known process in contrast to the intracellular cholesterol transport, which is poorly understood. Recently in our laboratory, we identified a novel protein in C. elegans involved in dietary cholesterol uptake, which we have named ChUP-1. Insillicoanalysis identified two putative orthologue candidate proteins in mammals. The proteins SIDT1 and SIDT2 share identity and conserved cholesterol binding (CRAC) domains with C. elegans ChUP-1. Both mammalian proteins are annotated as RNA transporters in databases. In the present study, we show evidence indicating that SIDT1 and SIDT2 not only do not transport RNA, but they are involved in cholesterol transport. Furthermore, we show that single point mutations directed to disrupt the CRAC domains of both proteins prevent FRET between SIDT1 and SIDT2 and the cholesterol analogue dehydroergosterol (DHE) and alter cholesterol transport.

Association of the IP3R to STIM1 provides a reduced intraluminal calcium microenvironment, resulting in enhanced store-operated calcium entry

The involvement of inositol trisphosphate receptor (IP3R) in modulating store-operated calcium entry (SOCE) was established many years ago. Nevertheless, the molecular mechanism responsible for this observation has not been elucidated to this date. In the present study we show that IP3R associates to STIM1 upon depletion of the endoplasmic reticulum (ER) by activation of the inositol trisphosphate signaling cascade via G-protein coupled receptors. IP3R-STIM1 association results in enhanced STIM1 puncta formation and larger Orai-mediated whole-cell currents as well as increased calcium influx. Depleting the ER with a calcium ATPase inhibitor (thapsigargin, TG) does not induce IP3R-STIM1 association, indicating that this association requires an active IP3R. The IP3R-STIM1 association is only observed after IP3R activation, as evidenced by FRET experiments and co-immunoprecipitation assays. ER intraluminal calcium measurements using Mag-Fluo-4 showed enhanced calcium depletion when IP3R is overexpressed. A STIM1-GCaMP fusion protein indicates that STIM1 detects lower calcium concentrations near its EF-hand domain when IP3R is overexpressed when compared with the fluorescence reported by a GCaMP homogenously distributed in the ER lumen (ER-GCaMP). All these data together strongly suggest that activation of inositol trisphosphate signaling cascade induces the formation of the IP3R-STIM1 complex. The activated IP3R provides a reduced intraluminal calcium microenvironment near STIM1, resulting in enhanced activation of Orai currents and SOCE.

Single-Channel Single-Molecule Detection (SC-SMD) System

The combination of single-molecule fluorescence imaging and electrophysiology provides a powerful tool to explore the stoichiometry and functional properties of ionic channels, simultaneously. Here, we describe a typical SC-SMD experiment from the preparation of plasmids containing the genes encoding for the channels of interest fused to fluorescent proteins to the use of the SC-SMD system for simultaneous patch clamping and single-molecule determinations.