Ca-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.

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

Unlabelled: 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.

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.

Identification of β-synuclein on secretory granules in chromaffin cells and the effects of α- and β-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-, β-, and γ-synuclein contains six to seven 11-amino acid repeats that are predicted to form amphipathic helices.

A pUL25 dimer interfaces the pseudorabies virus capsid and tegument.

Inside the virions of α-herpesviruses, tegument protein pUL25 anchors the tegument to capsid vertices through direct interactions with tegument proteins pUL17 and pUL36. In addition to promoting virion assembly, both pUL25 and pUL36 are critical for intracellular microtubule-dependent capsid transport. Despite these essential roles during infection, the stoichiometry and precise organization of pUL25 and pUL36 on the capsid surface remain controversial due to the insufficient resolution of existing reconstructions from cryo-electron microscopy (cryoEM).

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.

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).

Differential protein partitioning within the herpesvirus tegument and envelope underlies a complex and variable virion architecture.

The herpesvirus virion is a multilayered structure consisting of a DNA-filled capsid, tegument, and envelope. Detailed reconstructions of the capsid are possible based on its icosahedral symmetry, but the surrounding tegument and envelope layers lack regular architecture. To circumvent limitations of symmetry-based ultrastructural reconstruction methods, a fluorescence approach was developed using single-particle imaging combined with displacement measurements at nanoscale resolution.

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.

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.

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.

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.

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.