Deciphering interplay between Salmonella invasion effectors

Bacterial pathogens have evolved a specialized type III secretion system (T3SS) to translocate virulence effector proteins directly into eukaryotic target cells. Salmonellae deploy effectors that trigger localized actin reorganization to force their own entry into non-phagocytic host cells. Six effectors (SipC, SipA, SopE/2, SopB, SptP) can individually manipulate actin dynamics at the plasma membrane, which acts as a ‘signaling hub’ during Salmonella invasion. The extent of crosstalk between these spatially coincident effectors remains unknown. Here we describe trans and cis binary entry effector interplay (BENEFIT) screens that systematically examine functional associations between effectors following their delivery into the host cell. The results reveal extensive ordered synergistic and antagonistic relationships and their relative potency, and illuminate an unexpectedly sophisticated signaling network evolved through longstanding pathogen–host interaction

The zipper mechanism in phagocytosis: energetic requirements and variability in phagocytic cup shape

Phagocytosis is the fundamental cellular process by which eukaryotic cells bind and engulf particles by their cell membrane. Particle engulfment involves particle recognition by cell-surface receptors, signaling and remodeling of the actin cytoskeleton to guide the membrane around the particle in a zipper-like fashion. Despite the signaling complexity, phagocytosis also depends strongly on biophysical parameters, such as particle shape, and the need for actin-driven force generation remains poorly understood. Here, we propose a novel, three-dimensional and stochastic biophysical model of phagocytosis, and study the engulfment of particles of various sizes and shapes, including spiral and rod-shaped particles reminiscent of bacteria. Highly curved shapes are not taken up, in line with recent experimental results. Furthermore, we surprisingly find that even without actin-driven force generation, engulfment proceeds in a large regime of parameter values, albeit more slowly and with highly variable phagocytic cups. We experimentally confirm these predictions using fibroblasts, transfected with immunoreceptor FcyRIIa for engulfment of immunoglobulin G-opsonized particles. Specifically, we compare the wild-type receptor with a mutant receptor, unable to signal to the actin cytoskeleton. Based on the reconstruction of phagocytic cups from imaging data, we indeed show that cells are able to engulf small particles even without support from biological actin-driven processes. This suggests that biochemical pathways render the evolutionary ancient process of phagocytic highly robust, allowing cells to engulf even very large particles. The particle-shape dependence of phagocytosis makes a systematic investigation of host-pathogen interactions and an efficient design of a vehicle for drug delivery possible.Comment: Accepted for publication in BMC Systems Biology. 17 pages, 6 Figures, + supplementary informatio

Phosphorylation of nephrin induces phase separated domains that move through actomyosin contraction

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Kim, S., Kalappurakkal, J. M., Mayor, S., & Rosen, M. K. Phosphorylation of nephrin induces phase separated domains that move through actomyosin contraction. Molecular Biology of the Cell, 30(24), (2019): 2996–3012, doi:10.1091/mbc.E18-12-0823.The plasma membrane of eukaryotic cells is organized into lipid and protein microdomains, whose assembly mechanisms and functions are incompletely understood. We demonstrate that proteins in the nephrin/Nck/N-WASP actin-regulatory pathway cluster into micron-scale domains at the basal plasma membrane upon triggered phosphorylation of transmembrane protein nephrin. The domains are persistent but readily exchange components with their surroundings, and their formation is dependent on the number of Nck SH3 domains, suggesting they are phase separated polymers assembled through multivalent interactions among the three proteins. The domains form independent of the actin cytoskeleton, but acto-myosin contractility induces their rapid lateral movement. Nephrin phosphorylation induces larger clusters at the cell periphery, which are associated with extensive actin assembly and dense filopodia. Our studies illustrate how multivalent interactions between proteins at the plasma membrane can produce micron-scale organization of signaling molecules, and how the resulting clusters can both respond to and control the actin cytoskeleton.We thank Hongtao Yu (University of Texas Southwestern Medical Center [UTSW]) for providing the HeLa cell line used in this work; Dan Billadeau and Timothy Gomez (Mayo Clinic) for providing antibodies; Nico Stuurman (University of California, San Francisco) for assistance with STORM imaging; Kate Luby-Phelps and Abhijit Bugde (UTSW Live Cell Imaging Core Facility) for their assistance in epifluorescence and spinning disk confocal experiments; Sudeep Banjade for advice on designing the S3, S2, S1 constructs; Khuloud Jaqaman (UTSW) for advice on cluster motility analysis; Salman Banani and Jonathan Ditlev (UTSW) for critical reading of the manuscript; and members of the Rosen lab and participants in the MBL/HHMI Summer Institutes for advice and helpful discussions. This work was supported by a Howard Hughes Medical Institute Collaborative Innovation Award; the Welch Foundation (I-1544 to M.K.R.); a J.C. Bose Fellowship from the Department of Science and Technology, government of India (to S.M.); a Margadarshi Fellowship from the Wellcome Trust—Department of Biotechnology, India Alliance (IA/M/15/1/502018 to S.M.). Research in the Rosen lab is supported by the Howard Hughes Medical Institute

Ultrastructural and functional fate of recycled vesicles in hippocampal synapses

Efficient recycling of synaptic vesicles is thought to be critical for sustained information transfer at central terminals. However, the specific contribution that retrieved vesicles make to future transmission events remains unclear. Here we exploit fluorescence and time-stamped electron microscopy to track the functional and positional fate of vesicles endocytosed after readily releasable pool (RRP) stimulation in rat hippocampal synapses. We show that most vesicles are recovered near the active zone but subsequently take up random positions in the cluster, without preferential bias for future use. These vesicles non-selectively queue, advancing towards the release site with further stimulation in an actin-dependent manner. Nonetheless, the small subset of vesicles retrieved recently in the stimulus train persist nearer the active zone and exhibit more privileged use in the next RRP. Our findings reveal heterogeneity in vesicle fate based on nanoscale position and timing rules, providing new insights into the origins of future pool constitution

Cholesterol impairment contributes to neuroserpin aggregation

Intraneural accumulation of misfolded proteins is a common feature of several neurodegenerative pathologies including Alzheimer's and Parkinson's diseases, and Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB). FENIB is a rare disease due to a point mutation in neuroserpin which accelerates protein aggregation in the endoplasmic reticulum (ER). Here we show that cholesterol depletion induced either by prolonged exposure to statins or by inhibiting the sterol regulatory binding-element protein (SREBP) pathway also enhances aggregation of neuroserpin proteins. These findings can be explained considering a computational model of protein aggregation under non-equilibrium conditions, where a decrease in the rate of protein clearance improves aggregation. Decreasing cholesterol in cell membranes affects their biophysical properties, including their ability to form the vesicles needed for protein clearance, as we illustrate by a simple mathematical model. Taken together, these results suggest that cholesterol reduction induces neuroserpin aggregation, even in absence of specific neuroserpin mutations. The new mechanism we uncover could be relevant also for other neurodegenerative diseases associated with protein aggregation.Comment: 7 figure

Emulsion Inks: A New Class of Materials for 3D Printing Porous Tissue Engineered Grafts

Tissue grafts are often crucial in restoring function and promoting healing after traumatic injury. Many synthetic materials have been developed, but these often suffer from inadequate tissue integration, limited biodegradability, and mechanical mismatch with the target tissue. Recent advances in 3D printing technologies have enabled the fabrication of custom-fit scaffolds that resemble native tissue. Although these scaffolds can more closely mimic defect shape, new inks are needed to provide tunable control over multiple levels of scaffold structure and function. To address these limitations, we have developed an extensible system for printing complex tissue engineered scaffolds by creating emulsion templated inks. These emulsion inks exhibit tunable pore sizes, modulus, and strength. Formulation of inks with viscous, reactive macromers results in extruded material that holds its shape after extrusion and polymerizes rapidly upon exposure to UV light. New methodology was developed to permit the rational design of emulsion inks based on rheological and cure properties, and these inks were able to successfully create high fidelity scaffolds with customizable, hierarchical porosity. Emulsion inks are compatible with nearly any hydrophobic macromer allowing development of inks with limitless chemical and material properties. Next, a hybrid printing system was developed for extrusion of thermoplastic PCL and PLA along with emulsion inks to provide mechanical reinforcement. Scaffolds without reinforcement exhibited an increase in permeability with a decrease in infill density, with detriment to their modulus and strength. Mechanical reinforcement with PLA, however, resulted in a significant increase in modulus and strength in all cases. The creation of novel emulsion inks from existing biomaterial systems opens the door to the creation of scaffolds with a wide range of physical and chemical properties. Finally, this system was extended to oil-in-water emulsions, termed hydrocolloid inks, to facilitate printing of hydrogels. Due to their low viscosity, high fidelity printing of hydrogels has typically been limited to SLA methods. SFF printing of hydrogel scaffolds frequently relies on thickeners and additives, but we have refined the rheological properties without modification of the hydrogel makeup by emulsifying with innocuous mineral oil. These 3D printed hydrogel scaffolds represent some of the highest fidelity reproductions of complex anatomical geometries in the literature to date. Additionally, this system provides a methodology for creating hydrocolloid inks from nearly any hydrogel biomaterial. In summary, we have developed a library of porous materials that can be used to improve tissue regeneration. Furthermore, the emulsion structure-property relationships explored here can be used in designing future emulsion inks. A combinatorial approach of tuning the ink and fabrication system allows for creation of complex scaffolds with improved biomimicry, allowing for a new generation of hierarchically porous tissue engineered constructs

Autophagy and the Liver

Autophagy is a cellular process that involves lysosomal degradation and recycling of intracellular organelles and proteins to maintain energy homeostasis during times of cellular stress [1]. It also serves to remove damaged cellular components such as mitochondria and long-lived proteins. Autophagy is catabolic mechanism and although hepatic autophagy performs the standard functions of degrading damaged organelles/aggregated proteins and regulating cell death it also regulates lipid accumulation within the liver. Autophagy can be divided into three distinct sub-groups that are discussed below. This chapter focuses upon the role of autophagy in a variety of liver diseases including hepatocellular carcinoma (HCC) and viral hepatitis. The increased understanding of the cellular machinery regulating autophagy within the liver may foster the development of therapeutic strategies that will ultimately help treat liver disease

Actin assembly ruptures the nuclear envelope by prying the lamina away from nuclear pores and nuclear membranes in starfish oocytes.

The nucleus of oocytes (germinal vesicle) is unusually large and its nuclear envelope (NE) is densely packed with nuclear pore complexes (NPCs) stockpiled for embryonic development. We showed that breakdown of this specialized NE is mediated by an Arp2/3-nucleated F-actin 'shell' in starfish oocytes, in contrast to microtubule-driven tearing in mammalian fibroblasts. Here, we address the mechanism of F-actin-driven NE rupture by correlated live-cell, super-resolution and electron microscopy. We show that actin is nucleated within the lamina sprouting filopodia-like spikes towards the nuclear membranes. These F-actin spikes protrude pore-free nuclear membranes, whereas the adjoining membrane stretches accumulate NPCs associated with the still-intact lamina. Packed NPCs sort into a distinct membrane network, while breaks appear in ER-like, pore-free regions. Thereby, we reveal a new function for actin-mediated membrane shaping in nuclear rupture that is likely to have implications in other contexts such as nuclear rupture observed in cancer cells

Mechanically induced homochirality in nucleated enantioselective polymerization

Understanding how biological homochirality may have emerged during chemical evolution remains a challenge for origin of life research. In keeping with this goal, we introduce and solve numerically a kinetic rate equation model of nucleated cooperative enantioselective polymerization in closed systems. The microreversible scheme includes (i) solution phase racemization of the monomers, (ii) linear chain growth by stepwise monomer attachment, in both the nucleation and elongation phases, and (iii) annealing or fusion of homochiral chains. Mechanically induced breakage of the longest chains maintains the system out of equilibrium and drives a breakage-fusion recycling mechanism. Spontaneous mirror symmetry breaking (SMSB) can be achieved starting from small initial enantiomeric excesses due to the intrinsic statistical fluctuations about the idealized racemic composition. The subsequent chiral amplification confirms the model’s capacity for absolute asymmetric synthesis, and without chiral cross-inhibition and without explicit autocatalysis