Ca2+-sensor ALG-2 engages ESCRTs to enhance lysosomal membrane resilience to osmotic stress.

Lysosomes are central players in cellular catabolism, signaling, and metabolic regulation. Cellular and environmental stresses that damage lysosomal membranes can compromise their function and release toxic content into the cytoplasm. Here, we examine how cells respond to osmotic stress within lysosomes. Using sensitive assays of lysosomal leakage and rupture, we examine acute effects of the osmotic disruptant glycyl-L-phenylalanine 2-naphthylamide (GPN). Our findings reveal that low concentrations of GPN rupture a small fraction of lysosomes, but surprisingly trigger Ca2+ release from nearly all. Chelating cytoplasmic Ca2+ makes lysosomes more sensitive to GPN-induced rupture, suggesting a role for Ca2+ in lysosomal membrane resilience. GPN-elicited Ca2+ release causes the Ca2+-sensor Apoptosis Linked Gene-2 (ALG-2), along with Endosomal Sorting Complex Required for Transport (ESCRT) proteins it interacts with, to redistribute onto lysosomes. Functionally, ALG-2, but not its ESCRT binding-disabled ΔGF122 splice variant, increases lysosomal resilience to osmotic stress. Importantly, elevating juxta-lysosomal Ca2+ without membrane damage by activating TRPML1 also recruits ALG-2 and ESCRTs, protecting lysosomes from subsequent osmotic rupture. These findings reveal that Ca2+, through ALG-2, helps bring ESCRTs to lysosomes to enhance their resilience and maintain organelle integrity in the face of osmotic stress.

Refractive Index Imaging of Cells with Variable-Angle Near-Total Internal Reflection (TIR) Microscopy.

The refractive index in the interior of single cells affects the evanescent field depth in quantitative studies using total internal reflection (TIR) fluorescence, but often that index is not well known. We here present method to measure and spatially map the absolute index of refraction in a microscopic sample, by imaging a collimated light beam reflected from the substrate/buffer/cell interference at variable angles of incidence. Above the TIR critical angle (which is a strong function of refractive index), the reflection is 100%, but in the immediate sub-critical angle zone, the reflection intensity is a very strong ascending function of incidence angle. By analyzing the angular position of that edge at each location in the field of view, the local refractive index can be estimated. In addition, by analyzing the steepness of the edge, the distance-to-substrate can be determined. We apply the technique to liquid calibration samples, silica beads, cultured Chinese hamster ovary cells, and primary culture chromaffin cells. The optical technique suffers from decremented lateral resolution, scattering, and interference artifacts. However, it still provides reasonable results for both refractive index (~1.38) and for distance-to-substrate (~150 nm) for the cells, as well as a lateral resolution to about 1 µm.

Ca 2+ -sensor ALG-2 engages ESCRTs to enhance lysosomal membrane resilience to osmotic stress.

Lysosomes are central players in cellular catabolism, signaling, and metabolic regulation. Cellular and environmental stresses that damage lysosomal membranes can compromise their function and release toxic content into the cytoplasm. Here, we examine how cells respond to osmotic stress within lysosomes. Using sensitive assays of lysosomal leakage and rupture, we examine acute effects of the cathepsin C-metabolized osmotic disruptant glycyl-L-phenylalanine 2-naphthylamide (GPN). Our findings reveal that widely used concentrations of GPN rupture only a small fraction of lysosomes, but surprisingly trigger Ca 2+ release from nearly all. Chelating cytoplasmic Ca 2+ using BAPTA makes lysosomes more likely to rupture under GPN-induced stress, suggesting that Ca 2+ plays a role in protecting or rapidly repairing lysosomal membranes. Mechanistically, we establish that GPN causes the Ca 2+ -sensitive protein Apoptosis Linked Gene-2 (ALG-2) and interacting ESCRT proteins to redistribute onto lysosomes, improving their resistance to membrane stress created by GPN as well as the lysosomotropic drug chlorpromazine. Furthermore, we show that activating the cation channel TRPML1, with or without blocking the endoplasmic reticulum Ca 2+ pump, creates local Ca 2+ signals that protect lysosomes from rupture by recruiting ALG-2 and ESCRTs without any membrane damage. These findings reveal that Ca 2+ , through ALG-2, helps bring ESCRTs to lysosomes to enhance their resilience and maintain organelle integrity in the face of osmotic stress. SIGNIFICANCE: As the degradative hub of the cell, lysosomes are full of toxic content that can spill into the cytoplasm. There has been much recent interest in how cells sense and repair lysosomal membrane damage using ESCRTs and cholesterol to rapidly fix "nanoscale damage". Here, we extend understanding of how ESCRTs contribute by uncovering a preventative role of the ESCRT machinery. We show that ESCRTs, when recruited by the Ca 2+ -sensor ALG-2, play a critical role in stabilizing the lysosomal membrane against osmotically-induced rupture. This finding suggests that cells have mechanisms not just for repairing but also for actively protecting lysosomes from stress-induced membrane damage.

Fusion of a fluorescent protein to the pUL25 minor capsid protein of pseudorabies virus allows live-cell capsid imaging with negligible impact on infection.

In order to resolve the location and activity of submicroscopic viruses in living cells, viral proteins are often fused to fluorescent proteins (FPs) and visualized by microscopy. In this study, we describe the fusion of FPs to three proteins of pseudorabies virus (PRV) that allowed imaging of capsids in living cells. Included in this study are the first recombinant PRV strains expressing FP-pUL25 fusions based on a design applied to herpes simplex virus type 1 by Homa and colleagues. The properties of each reporter virus were compared in both in vitro and in vivo infection models. PRV strains expressing FP-pUL25 and FP-pUL36 preserved wild-type properties better than traditional FP-pUL35 isolates in assays of plaque size and virulence in mice. The utility of these strains in studies of axon transport, nuclear dynamics and viral particle composition are documented.

ESCRT puts its thumb on the nanoscale: Fixing tiny holes in endolysosomes.

The ESCRT (endosomal complex required for transport) machinery remodels membranes to bud vesicles away from the cytoplasm. In addition to this classic role, ESCRTs are now understood to repair damage in the plasma membrane, nuclear envelope, and throughout the endolysosomal network. Wounds in endolysosomal membranes are caused by pathogens, particulates, and other chemical or metabolic stresses. Nanoscale damage in these membranes promotes activation and engagement of ESCRT proteins. A full understanding of damage signals, molecular sensing, and the mechanism of membrane repair is yet to be developed. Nevertheless, a triggering role for calcium and ESCRT-I in recruiting ESCRT-III machinery for membrane remodeling is a repeated theme in functional studies of this response. In our current understanding of the continuum of cellular responses to lipid bilayer damage, the ESCRT machinery is fast, sensitive, and deployed independently of other systems.

Identification of ß-synuclein on secretory granules in chromaffin cells and the effects of α- and ß-synuclein on post-fusion BDNF discharge and fusion pore expansion.

α-Synuclein is strongly implicated in the pathogenesis of Parkinson's disease as well as in other neurodegenerative diseases. However, its normal function in cells is not understood. The N-termini of α-, ß-, and γ-synuclein contains six to seven 11-amino acid repeats that are predicted to form amphipathic helices. Membrane-binding and membrane-curving abilities of synuclein raise the possibility that synuclein could alter cellular processes that involve highly curved structures. In the present study we examined the localization of endogenous synuclein in bovine chromaffin cells by immunocytochemistry and its possible function to control protein discharge upon fusion of the granule with the plasma membrane by regulating the fusion pore. We found with quantitative immunocytochemistry that endogenous ß-synuclein associates with secretory granules. Endogenous α-synuclein only rarely co-localizes with secretory granules. Overexpression of α-synuclein but not ß-synuclein quickened the post- fusion discharge of BDNF-pHluorin by approxinately 30%. However, neither α- nor ß-synuclein significantly altered curvature dynamics associated with fusion pore expansion that were measured by the combination of polarization and total internal reflection fluorescence microscopy (pTIRFM). Whatever the mechanism, the physiological significance of the small increased rate of post-fusion protein discharge caused by α-synuclein remains to be demonstrated, especially since endogenous ß-, but not α-synuclein is the predominant synuclein isoform associated with chromaffin granules.

The synaptotagmin C2B domain calcium-binding loops modulate the rate of fusion pore expansion.

In chromaffin cells, the kinetics of fusion pore expansion vary depending on which synaptotagmin isoform (Syt-1 or Syt-7) drives release. Our recent studies have shown that fusion pores of granules harboring Syt-1 expand more rapidly than those harboring Syt-7. Here we sought to define the structural specificity of synaptotagmin action at the fusion pore by manipulating the Ca2+-binding C2B module. We generated a chimeric Syt-1 in which its C2B Ca2+-binding loops had been exchanged for those of Syt-7. Fusion pores of granules harboring a Syt-1 C2B chimera with all three Ca2+-binding loops of Syt-7 (Syt-1:7C2B123) exhibited slower rates of fusion pore expansion and neuropeptide cargo release relative to WT Syt-1. After fusion, this chimera also dispersed more slowly from fusion sites than WT protein. We speculate that the Syt-1:7 C2B123 and WT Syt-1 are likely to differ in their interactions with Ca2+ and membranes. Subsequent in vitro and in silico data demonstrated that the chimera exhibits a higher affinity for phospholipids than WT Syt-1. We conclude that the affinity of synaptotagmin for the plasma membrane, and the rate at which it releases the membrane, contribute in important ways to the rate of fusion pore expansion.

Slow fusion pore expansion creates a unique reaction chamber for co-packaged cargo.

A lumenal secretory granule protein, tissue plasminogen activator (tPA), greatly slows fusion pore dilation and thereby slows its own discharge. We investigated another outcome of the long-lived narrow fusion pore: the creation of a nanoscale chemical reaction chamber for granule contents in which the pH is suddenly neutralized upon fusion. Bovine adrenal chromaffin cells endogenously express both tPA and its primary protein inhibitor, plasminogen activator inhibitor 1 (PAI). We found by immunocytochemistry that tPA and PAI are co-packaged in the same secretory granule. It is known that PAI irreversibly and covalently inactivates tPA at neutral pH. We demonstrate with zymography that the acidic granule lumen protects tPA from inactivation by PAI. Immunocytochemistry, total internal reflection fluorescence (TIRF) microscopy, and polarized TIRF microscopy demonstrated that co-packaged PAI and tPA remain together in granules for many seconds in the nanoscale reaction chamber, more than enough time to inhibit tPA and create a new secreted protein species.

Drosophila Schneider 2 (S2) cells: a novel tool for studying HSV-induced membrane fusion.

Drosophila S2 cells and mammalian CHO-K1 cells were used to investigate the requirements for HSV-1 cell fusion. Infection assays indicated S2 cells were not permissive for HSV-1. HVEM and nectin-1 mediated cell fusion between CHO-K1 cells and S2 cells when either CHO-K1 or S2 cells were used as target cells. Interestingly, PILRα did not mediate fusion between CHO-K1 or S2 cells due to a glycosylation defect of PILRα and gB in S2 cells. Fusion activity was not detected for any receptor tested when S2 cells were used both as target cells and effector cells indicating S2 cells may lack a key cellular factor present in mammalian cells that is required for cell fusion. Thus, insect cells may provide a novel tool to study the interaction of HSV-1 glycoproteins and cellular factors required for fusion, as well as a means to identify unknown cellular factors required for HSV replication.

The herpesvirus VP1/2 protein is an effector of dynein-mediated capsid transport and neuroinvasion.

Microtubule transport of herpesvirus capsids from the cell periphery to the nucleus is imperative for viral replication and, in the case of many alphaherpesviruses, transmission into the nervous system. Using the neuroinvasive herpesvirus, pseudorabies virus (PRV), we show that the viral protein 1/2 (VP1/2) tegument protein associates with the dynein/dynactin microtubule motor complex and promotes retrograde microtubule transport of PRV capsids. Functional activation of VP1/2 requires binding to the capsid protein pUL25 or removal of the capsid-binding domain. A proline-rich sequence within VP1/2 is required for the efficient interaction with the dynein/dynactin microtubule motor complex as well as for PRV virulence and retrograde axon transport in vivo. Additionally, in the absence of infection, functionally active VP1/2 is sufficient to move large surrogate cargoes via the dynein/dynactin microtubule motor complex. Thus, VP1/2 tethers PRV capsids to dynein/dynactin to enhance microtubule transport, neuroinvasion, and pathogenesis.

Alphaherpesviruses and the cytoskeleton in neuronal infections.

Following infection of exposed peripheral tissues, neurotropic alphaherpesviruses invade nerve endings and deposit their DNA genomes into the nuclei of neurons resident in ganglia of the peripheral nervous system. The end result of these events is the establishment of a life-long latent infection. Neuroinvasion typically requires efficient viral transmission through a polarized epithelium followed by long-distance transport through the viscous axoplasm. These events are mediated by the recruitment of the cellular microtubule motor proteins to the intracellular viral particle and by alterations to the cytoskeletal architecture. The focus of this review is the interplay between neurotropic herpesviruses and the cytoskeleton.

MCAK and paclitaxel have differential effects on spindle microtubule organization and dynamics.

Within the mitotic spindle, there are multiple populations of microtubules with different turnover dynamics, but how these different dynamics are maintained is not fully understood. MCAK is a member of the kinesin-13 family of microtubule-destabilizing enzymes that is required for proper establishment and maintenance of the spindle. Using quantitative immunofluorescence and fluorescence recovery after photobleaching, we compared the differences in spindle organization caused by global suppression of microtubule dynamics, by treating cells with low levels of paclitaxel, versus specific perturbation of spindle microtubule subsets by MCAK inhibition. Paclitaxel treatment caused a disruption in spindle microtubule organization marked by a significant increase in microtubules near the poles and a reduction in K-fiber fluorescence intensity. This was correlated with a faster t(1/2) of both spindle and K-fiber microtubules. In contrast, MCAK inhibition caused a dramatic reorganization of spindle microtubules with a significant increase in astral microtubules and reduction in K-fiber fluorescence intensity, which correlated with a slower t(1/2) of K-fibers but no change in the t(1/2) of spindle microtubules. Our data support the model that MCAK perturbs spindle organization by acting preferentially on a subset of microtubules, and they support the overall hypothesis that microtubule dynamics is differentially regulated in the spindle.