Biophysics of the Cytoskeletal- Translationally and Immediately Post-Translationally
Total Page：16
File Type：pdf, Size：1020Kb
Sunday, February 12, 2017 15a Symposium: Proteins in Vivo: From the external amyloid aggregates rapidly surround the cells and act as a recruitment point for soluble protein, triggering amyloid fibril elongation. A fraction of Ribosome Through the Chaperone to the Native aggregates surrounding the cardiomyocytes is internalized via micropinocyto- State sis (same mechanism followed by soluble proteins). AL amyloid fibrils are shown to cause cell growth arrest at low concentration. Soluble proteins induce 80-Symp apoptosis, demonstrating different cytotoxic mechanisms between soluble pro- Channeling Nascent Proteins Towards the Native State: Role of the tein and amyloid aggregates. Ribosome and Molecular Chaperones In vitro amyloid formation experiments show that heterologous recruitment of Silvia Cavagnero, Rayna M. Addabbo, Matthew D. Dalphin, Yue Liu, light chains is kinetically determined by the conformation of the amyloidogenic Miranda F. Mecha. conformational precursor and modulated by the differential ability of each pro- Chemistry, University of Wisconsin-Madison, Madison, WI, USA. tein to either nucleate or elongate fibrils. Despite much progress over the last six decades, we are still far from understand- Studies on the early events of fibril formation followed by dynamic light scat- ing the mechanism of protein folding and aggregation in the cell. This lack of in- tering, chromatography and electron microscopy have shown differences in the formation poses tremendous challenges to progress in many areas of life sciences, species formed by amyloidogenic proteins (stable small oligomers) compared and it severely impedes key efforts in biomedical research. Learning more about to control proteins (large oligomers observed). the interplay between protein folding and aggregation in bacterial cells has a direct Overall, our studies emphasize the complex interactions between light chain impact on the development of strategies to treat microbial infection and on the and cells that result in fibril internalization, protein recruitment, and cytotox- optimization of protein-based drug production in the pharmaceutical industry. icity that may occur in AL amyloidosis. In bacteria, the majority of soluble cellular proteins fold or aggregate co- Symposium: Biophysics of the Cytoskeletal- translationally and immediately post-translationally. The ribosome and molecular chaperones play a key role in this process. This presentation will report progress, Membrane Interface failures and surprises on our molecular-level understanding of how the ribosome, ribosomal proteins and cotranslationally active molecular chaperones modulate 83-Symp the balance between protein folding and aggregation in the cell. By probing the Modeling Membrane Tubules with Lipid Droplets and Migrasomes structure and dynamics of nascent proteins by fluorescence depolarization in the Michael M. Kozlov. frequency domain, multidimensional NMR, biochemical tools and kinetic simu- Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv lations, we will attempt to recapitulate some fundamental concepts of general sig- University, Tel Aviv, Israel. nificance, and suggest how these concepts can be specifically exploited to Membrane tubules of few tens of nanometer cross-sectional diameters and understand and reprogram the bacterial translation machinery to maximize the micron-scale lengths represent a basic structural component of intra-cellular yield and timely production of correctly folded proteins. organelles, such as endoplasmic reticulum and Golgi Complex, and emerge from plasma membranes in the course of cell crawling on extra-cellular 81-Symp matrices and substrates. Besides barrier functions, the tubular membranes Ribosomes in Motion: The Dynamics of Nature’s Protein Synthesis serve as platforms for formation of peculiar cell organelles, Lipid Droplets Machinery and Migrasomes, whose properties are to be understood in terms of simple Ruben L. Gonzalez Jr. physics. Lipid Droplet can be regarded as lenses of hydrophobic substance Department of Chemistry, Columbia University, New York, NY, USA. (triacylglycerol, sterol esters) growing up between the two membrane leaflets The ribosome can be regarded as a molecular machine that converts chemical and into micron-large buds, which, possibly, detach from the membrane to form thermal energy into productive mechanical work. This chemo- and thermome- emulsion-like droplets. Migrasomes are micron-size spherical bodies discov- chanical view of ribosome function continues to fuel efforts to identify the mobile ered, recently, to form on membrane tethers pulled out of the cell body in components of the ribosomal machine, characterize the structural dynamics of the course of cell migration. We address the micromechanics of the two organ- these components, and develop an understanding of how these dynamics are regu- elles to gain understanding of physics behind their formation and evolution. lated in order to direct mechanical processes during protein synthesis. It is within We analyze the shape and energy of a membrane tubule containing a lipid this context that our research group has developed a fully reconstituted, fluores- droplet in dependence of the droplet size and the elastic properties of the cently labeled in vitro translation system and used it for single-molecule fluores- tubular membrane and the lipid monolayers covering the droplet surface. cence imaging studies of protein synthesis. Together with ensemble biochemical We determine the conditions of the droplet detachment from the tubule. We investigations of protein synthesis by the ribosome and structural studies of func- suggest a physical model of Migrasome as a rigid membrane domain, which tional ribosomal complexes, single-molecule fluorescence imaging of protein syn- swells within a membrane tubule into a sphere-like body as a result of interplay thesis continues to provide unique and powerful mechanistic insights into this between the membrane bending rigidity and lateral tension. By computing the fundamental biological process. In this talk, I will present recent and ongoing domain shapes for varying membrane elastic properties and the domain sizes work from our research group aimed at developing an ever-deeper understanding and comparing the results with the available images, we suggest the criteria of the function of the ribosome during protein synthesis. In addition, I will discuss of Migrasome formation. what we envision lies ahead as single-molecule fluorescence imaging approaches continue to evolve and expand to address increasingly complex mechanistic and 84-Symp regulatory aspects of this fundamental biological process. Multiscale Simulation of Proteins at the Membrane-Cytoskeleton Interface Gregory A. Voth. 82-Symp University of Chicago, Chicago, IL, USA. From Native to Amyloid in the Test Tube and in Cells: A Journey Protein-protein interactions at the cell membrane play a crucial role in dictating of Misbehaving Antibodies cell morphology, adhesion, motility, and cytokinesis. Examples include the Marina Ramirez-Alvarado1, Marta Marin-Argany1, Christopher J. Dick1, coupling of transmembrane integrins with the cytoskeleton as well as Luis M. Blancas-Mejia1, Pinaki Misra1, Yi Lin2, Angela Williams3, formin-driven polymerization of actin filaments in fission yeast ring assembly. Jonathan S. Wall3. The intrinsic mechanosensitivity of these proteins is an important determinant 1Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA, of emergent large-scale effects from collections of membrane-associated com- 2Medicine-Division of Hematology, Mayo Clinic, Rochester, MN, USA, plexes such as focal adhesions and cytokinetic nodes. Whether integrin and 3Medicine and Radiology, The University of Tennessee Graduate School of formin-nodes clustering respond to mechanical cues remains controversial in Medicine, Knoxville, TN, USA. the field, due to inherent limitations of experimental approaches in isolating Light chain (AL) amyloidosis is an incurable disease characterized by the specific protein contributions. Coarse-grained (CG) simulations of such sys- misfolding, aggregation, and systemic deposition of amyloid composed of tems can be indispensable tools for distinguishing between such competing sce- immunoglobulin light chains. We have conducted thermodynamic and fibril narios. In this talk, I will discuss how we apply CG simulations to recapitulate formation studies of both variable domain and full length immunoglobulin pro- the complex behavior observed by our experimental collaborators and place teins involved in AL amyloidosis. We have found that there is thermodynamic them into a rigorous framework to learn more about the microscopic behavior range -under physiological conditions- in which these proteins populate at play. First, using a new model of single-point, two state integrin particles on a partially folded states that favor amyloid formation. quasi-2D membrane, we demonstrate how cell spreading on soft substrates is We have studied the internalization of these same AL proteins in soluble form regulated by integrin activation and how multiple integrin subtypes differen- and as amyloid fibrils into human cardiomyocytes. Our results show how tially transmit traction stress. By further including explicit coupling to a BPJ 7693_7704 16a Sunday, February 12, 2017 fluctuating cytoskeleton, we have studied the effect of single actin filament and yiiP. CotP appears to have a role in Co2þ homeostasis as the deletion polymerization on integrin clustering