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S P Gross

Publications and source records attributed to S P Gross.

8 recordsLinked to original sources
PubMed2005

Building complexity: an in vitro study of cytoplasmic dynein with in vivo implications.

BACKGROUND: Cytoplasmic dynein is the molecular motor responsible for most retrograde microtubule-based vesicular transport. In vitro single-molecule experiments suggest that dynein function is not as robust as that of kinesin-1 or myosin-V because dynein moves only a limited distance (approximately 800 nm) before detaching and can exert a modest (approximately 1 pN) force. However, dynein-driven cargos in vivo move robustly over many microns and exert forces of multiple pN. To determine how to go from limited single-molecule function to robust in vivo transport, we began to build complexity in a controlled manner by using in vitro experiments. RESULTS: We show that a single cytoplasmic dynein motor frequently transitions into an off-pathway unproductive state that impairs net transport. Addition of a second (and/or third) dynein motor, so that cargos are moved by two (or three) motors rather than one, is sufficient to recover several properties of in vivo motion; such properties include long cargo travels, robust motion, and increased forces. Part of this improvement appears to arise from selective suppression of the unproductive state of dynein rather than from a fundamental change in dynein's mechanochemical cycle. CONCLUSIONS: Multiple dyneins working together suppress shortcomings of a single motor and generate robust motion under in vitro conditions. There appears to be no need for additional cofactors (e.g., dynactin) for this improvement. Because cargos are often driven by multiple dyneins in vivo, our results show that changing the number of dynein motors could allow modulation of dynein function from the mediocre single-dynein limit to robust in vivo-like dynein-driven motion.

Animals↗
PubMed2005

Heterogeneity of a fluorescent tegument component in single pseudorabies virus virions and enveloped axonal assemblies.

The molecular mechanisms responsible for long-distance, directional spread of alphaherpesvirus infections via axons of infected neurons are poorly understood. We describe the use of red and green fluorescent protein (GFP) fusions to capsid and tegument components, respectively, to visualize purified, single extracellular virions and axonal assemblies after pseudorabies virus (PRV) infection of cultured neurons. We observed heterogeneity in GFP fluorescence when GFP was fused to the tegument component VP22 in both single extracellular virions and discrete puncta in infected axons. This heterogeneity was observed in the presence or absence of a capsid structure detected by a fusion of monomeric red fluorescent protein to VP26. The similarity of the heterogeneous distribution of these fluorescent protein fusions in both purified virions and in axons suggested that tegument-capsid assembly and axonal targeting of viral components are linked. One possibility was that the assembly of extracellular and axonal particles containing the dually fluorescent fusion proteins occurred by the same process in the cell body. We tested this hypothesis by treating infected cultured neurons with brefeldin A, a potent inhibitor of herpesvirus maturation and secretion. Brefeldin A treatment disrupted the neuronal secretory pathway, affected fluorescent capsid and tegument transport in the cell body, and blocked subsequent entry into axons of capsid and tegument proteins. Electron microscopy demonstrated that in the absence of brefeldin A treatment, enveloped capsids entered axons, but in the presence of the inhibitor, unenveloped capsids accumulated in the cell body. These results support an assembly process in which PRV capsids acquire a membrane in the cell body prior to axonal entry and subsequent transport.

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PubMed2004

Local modulation of plus-end transport targets herpesvirus entry and egress in sensory axons.

The core structures of many viruses move within cells by association with host cytoskeletal motor proteins; however, the mechanisms by which intracellular viral particles are transported toward sites of replication or the cell periphery at distinct stages of infection remain to be understood. The regulation of herpesvirus directional transport in sensory neurons was examined by tracking individual viral capsids within axons at multiple frames per s. After entry into axons, capsids underwent bidirectional and saltatory movement to the cell body independently of endosomes. A comparison of entry transport to a previous analysis of capsid axonal transport during egress revealed that capsid targeting in and out of cells occurs by modulation of plus-end, but not minus-end, motion. Entry transport was unperturbed by the presence of egressing virus from a prior infection, indicating that transport direction is not modulated globally by viral gene expression, but rather directly by a component of the viral particle.

Animals↗
PubMed2001

Herpesviruses use bidirectional fast-axonal transport to spread in sensory neurons.

Alpha herpesviruses infect the vertebrate nervous system resulting in either mild recurrent lesions in mucosal epithelia or fatal encephalitis. Movement of virions within the nervous system is a critical factor in the outcome of infection; however, the dynamics of individual virion transport have never been assessed. Here we visualized and tracked individual viral capsids as they moved in axons away from infected neuronal cell bodies in culture. The observed movement was compatible with fast axonal flow mediated by multiple microtubule motors. Capsids accumulated at axon terminals, suggesting that spread from infected neurons required cell contact.

Animals↗
PubMed2000

Dynein-mediated cargo transport in vivo. A switch controls travel distance.

Cytoplasmic dynein is a microtubule-based motor with diverse cellular roles. Here, we use mutations in the dynein heavy chain gene to impair the motor's function, and employ biophysical measurements to demonstrate that cytoplasmic dynein is responsible for the minus end motion of bidirectionally moving lipid droplets in early Drosophila embryos. This analysis yields an estimate for the force that a single cytoplasmic dynein exerts in vivo (1.1 pN). It also allows us to quantitate dynein-mediated cargo motion in vivo, providing a framework for investigating how dynein's activity is controlled. We identify three distinct travel states whose general features also characterize plus end motion. These states are preserved in different developmental stages. We had previously provided evidence that for each travel direction, single droplets are moved by multiple motors of the same type (Welte et al. 1998). Droplet travel distances (runs) are much shorter than expected for multiple motors based on in vitro estimates of cytoplasmic dynein processivity. Therefore, we propose the existence of a process that ends runs before the motors fall off the microtubules. We find that this process acts with a constant probability per unit distance, and is typically coupled to a switch in travel direction. A process with similar properties governs plus end motion, and its regulation controls the net direction of transport.

Animals↗
PubMed1998

Developmental regulation of vesicle transport in Drosophila embryos: forces and kinetics.

In Drosophila embryos, microtubules oriented along apical-basal directions support saltatory vesicle movement. Vesicle traffic includes lipid droplets whose distribution shifts twice during early embryogenesis. Using microscopy, optical tweezers, and a novel squashed-mount embryo preparation, we tracked single droplets and measured the forces these generated. Droplet stalling forces change developmentally, in a roughly quantized fashion, consistent with variation in the number of active motors. We characterized a mutation, klarsicht, that affects droplet transport. Klar+ facilitates changes in force, possibly by coordinating the activity of multiple motors. Alterations in transport affected motion in both apical and basal directions, indicating tight coupling between motors of opposite polarity. Mutations in klar also affect nuclear migration during eye development, suggesting multiple roles for klar-based transport.

Animals↗
PubMed1984

Peritonitis with continuous ambulatory peritoneal dialysis and continuous cycling peritoneal dialysis.

The frequency and cause of peritonitis in 18 children receiving continuous ambulatory peritoneal dialysis (CAPD) and nine children receiving continuous cycling peritoneal dialysis (CCPD) are described. Cumulative CAPD and CCPD experience demonstrated 58 episodes of peritonitis in 294 patient treatment months (one case per 5.1 patient treatment months). Total hospitalization for the treatment of peritonitis was 0.18 days per patient treatment month. Life table analysis revealed no significant difference in the peritonitis-free "survival" between the two modalities. Gram-negative organisms accounted for a significantly increased percentage of the peritonitis in CAPD compared with CCPD (65% vs 17%) (P less than 0.001). Thirty-seven percent of the gram-negative infections in the CAPD population were in children with nephrostomies. Factors predisposing to peritonitis were identified in 76% of cases occurring with CAPD. Peritonitis remains the major contributor to the morbidity associated with peritoneal dialysis, regardless of the technique. The resultant frequency of hospitalization is not prohibitive. Attention to the "high-risk" pediatric patient and education directed at several well-recognized predisposing factors may yield improved results.

Acinetobacter Infections↗