Bulletin of the American Physical Society
APS March Meeting 2014
Volume 59, Number 1
Monday–Friday, March 3–7, 2014; Denver, Colorado
Session S3: Focus Session: Intracellular Organization |
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Sponsoring Units: DBIO Chair: Margaret Gardel, The University of Chicago Room: 107 |
Thursday, March 6, 2014 8:00AM - 8:12AM |
S3.00001: Self-organized spatiotemporal patterns of PIP$_{3}$ and PTEN during spontaneous cell polarization Fabian Knoch, Marco Tarantola, Wouter-Jan Rappel, Eberhard Bodenschatz During spontaneous cell polarization of Dictyostelium discoideum cells, PIP3 (phosphatidylinositol (3,4,5)-triphoshpate) and PTEN (phosphatase tensin homolog) have been identified as key signaling molecules, which govern the process of polarization in a self-organized manner. Gerisch et al. have shown that randomly triggered excitable PIP3 waves regulate the anti-correlated PTEN concentration. Here we show that this requires a switch-like dynamics of the overall membrane bound PTEN concentration in combination with two species of PTEN differing in their dephosphorylation rates. A quantitative modeling with a coupled reaction-diffusion system shows excellent agreement with experimental results and predicts a ratio $\sigma $ of dephosphorylation rates acting on PIP3 of $\sigma \approx $ 80 $-$ 100. Our quantitative analysis suggests that surface-attached cell membrane spanning PIP3 waves are necessary for resetting the global actin network. This is evidenced by the experimentally observed delay between polarization-cycles also quantitatively captured by our analysis. [Preview Abstract] |
Thursday, March 6, 2014 8:12AM - 8:24AM |
S3.00002: Dynamics of myosin II organization into cortical contractile networks and fibers Wei Nie, Ming-tzo Wei, Daniel Ou-Yang, Sabrina Jedlicka, Dimitrios Vavylonis The morphology of adhered cells critically depends on the formation of a contractile meshwork of parallel and cross-linked stress fibers along the contacting surface. The motor activity and mini-filament assembly of non-muscle myosin II is an important component of cell-level cytoskeletal remodeling during mechanosensing. To monitor the dynamics of myosin II, we used confocal microscopy to image cultured HeLa cells that stably express myosin regulatory light chain tagged with GFP (MRLC-GFP). MRLC-GFP was monitored in time-lapse movies at steady state and during the response of cells to varying concentrations of blebbistatin which disrupts actomyosin stress fibers. Using image correlation spectroscopy analysis, we quantified the kinetics of disassembly and reassembly of actomyosin networks and compared them to studies by other groups. This analysis suggested that the following processes contribute to the assembly of cortical actomyosin into fibers: random myosin mini-filament assembly and disassembly along the cortex; myosin mini-filament aligning and contraction; stabilization of cortical myosin upon increasing contractile tension. We developed simple numerical simulations that include those processes. The results of simulations of cells at steady state and in response to blebbistatin capture some of the main features observed in the experiments. This study provides a framework to help interpret how different cortical myosin remodeling kinetics may contribute to different cell shape and rigidity depending on substrate stiffness. [Preview Abstract] |
Thursday, March 6, 2014 8:24AM - 8:36AM |
S3.00003: Brownian dynamics simulation of fission yeast mitotic spindle formation Christopher Edelmaier, Robert A. Blackwell, Oliver M. Sweezy, Matthew A. Glaser, Meredith D. Betterton The mitotic spindle segregates chromosomes during mitosis. The dynamics that establish bipolar spindle formation are not well understood. We have developed a computational model of fission-yeast mitotic spindle formation using Brownian dynamics and kinetic Monte Carlo methods. Our model includes rigid, dynamic microtubules, a spherical nuclear envelope, spindle pole bodies anchored in the nuclear envelope, and crosslinkers and crosslinking motor proteins. Crosslinkers and crosslinking motor proteins attach and detach in a grand canonical ensemble, and exert forces and torques on the attached microtubules. We have modeled increased affinity for crosslinking motor attachment to antiparallel microtubule pairs, and stabilization of microtubules in the interpolar bundle. We study parameters controlling the stability of the interpolar bundle and assembly of a bipolar spindle from initially adjacent spindle-pole bodies. [Preview Abstract] |
Thursday, March 6, 2014 8:36AM - 9:12AM |
S3.00004: Membrane tension regulates clathrin-coated pit dynamics Invited Speaker: Allen Liu Intracellular organization depends on close communication between the extracellular environment and a network of cytoskeleton filaments. The interactions between cytoskeletal filaments and the plasma membrane lead to changes in membrane tension that in turns help regulate biological processes. Endocytosis is thought to be stimulated by low membrane tension and the removal of membrane increases membrane tension. While it is appreciated that the opposing effects of exocytosis and endocytosis have on keeping plasma membrane tension to a set point, it is not clear how membrane tension affects the dynamics of clathrin-coated pits (CCPs), the individual functional units of clathrin-mediated endocytosis. Furthermore, although it was recently shown that actin dynamics counteracts membrane tension during CCP formation, it is not clear what roles plasma membrane tension plays during CCP initiation. Based on the notion that plasma membrane tension is increased when the membrane area increases during cell spreading, we designed micro-patterned surfaces of different sizes to control the cell spreading sizes. Total internal reflection fluorescence microscopy of living cells and high content image analysis were used to quantify the dynamics of CCPs. We found that there is an increased proportion of CCPs with short (\textless 20s) lifetime for cells on larger patterns. Interestingly, cells on larger patterns have higher CCP initiation density, an effect unexpected based on the conventional view of decreasing endocytosis with increasing membrane tension. Furthermore, by analyzing the intensity profiles of CCPs that were longer-lived, we found CCP intensity decreases with increasing cell size, indicating that the CCPs are smaller with increasing membrane tension. Finally, disruption of actin dynamics significantly increased the number of short-lived CCPs, but also decreased CCP initiation rate. Together, our study reveals new mechanistic insights into how plasma membrane tension regulates the dynamics of CCPs. [Preview Abstract] |
Thursday, March 6, 2014 9:12AM - 9:24AM |
S3.00005: Probing the Effects of Cargo Loads in Single-Molecule Kinesin Mechanochemistry B.D. Jacobson, S.J. Koch, S.R. Atlas The influence of cargo loading on the dynamics of motor proteins such as kinesin is key to understanding fundamental aspects of their kinetic cycles and mechanochemistry. Kinetic models offer insight into these complex processes which occur on time scales up to seconds, and coupled with experimental data, they are a powerful tool in generating an increasingly fine-grained understanding of the chemical and mechanical mechanisms involved in kinesin procession, as well as the prospect of direct coupling to atomistic-scale simulations [1]. Here we present a kinetic model of single-molecule kinesin to study the effects of external forces due to intracellular cargo transport on chemical and mechanical rate constants. We use a simulated annealing algorithm that optimizes rate constants to fit published experimental data on kinesin speed and processivity, and kinetic Monte Carlo to compare predicted values with independent experimental measurements. We also discuss the application of sensitivity analysis to provide additional insight into the critical transitions and states of the processing protein under load.\\ \noindent[1] B. D. Jacobson, L. J. Herskowitz, S. J. Koch and S. R. Atlas, Investigation of kinesin processivity via simulated annealing, Biophys. J., submitted (2013). [Preview Abstract] |
Thursday, March 6, 2014 9:24AM - 9:36AM |
S3.00006: Deciphering Neurofilament Motility in live cells Christopher Johnson, Peter Jung Neurofilaments are the most abundant cytoskeletal elements of mature neuronal axons. They are assembled in the axon and responsible for regulating the axon's diameter. These filaments are transported in a characteristic stop-and-go fashion along microtubule tracks toward the nerve terminal driven by the motor proteins, kinesin and dynein.~ To explore the mechanisms underlying the observed stop-and-go transport, we devise a computational model~in which kinesin and dynein---coupled by respective force generation (tug-of-war)--- are attached to the neurofilament cargo.~ The resulting movement and kinetic characteristics are compared with highly time-resolved kymograph recordings.~ This comparison determines whether the observed kinetics are consistent with a tug-of-war model, and also renders an estimate of how many motors are attached to the cargo during transport. [Preview Abstract] |
Thursday, March 6, 2014 9:36AM - 10:12AM |
S3.00007: \textit{In vivo} control mechanisms of motor-cargo movement on microtubules Invited Speaker: Shermali Gunawardena Within axons, molecular motors transport essential components required for neuronal growth and viability. Although many levels of regulation must exist for proper anterograde and retrograde transport of vital proteins, little is known about these mechanisms. Previous work suggested that the amyloid precursor protein (APP) functions as a kinesin-1 receptor during transport. However, how APP vesicle motility is regulated is unclear. Using genetics and \textit{in vivo} imaging in \textit{Drosophila} we showed that reduction of presenilin (PS) substantially increased anterograde and retrograde APP vesicle velocities. Strikingly, PS deficiency had no effect on an unrelated cargo vesicle containing synaptotagmin, which is powered by a different kinesin motor. Increased PS-mediated velocities required functional kinesin-1 and dynein motors. We also found that these PS-mediated effects on motor protein function were mediated via a pathway that involves glycogen synthase kinase-3$\beta $ (GSK-3$\beta )$. PS genetically interacted with GSK-3$\beta $ in an activity dependent manner. Excess of active GSK-3$\beta $ perturbed transport by causing axonal blockages, which were enhanced by reduction of kinesin-1 or dynein, while excess of non-functional GSK-3$\beta $ had no effect. Strikingly, GSK-3$\beta $-activity dependent transport defects were enhanced by reduction of PS. Collectively, our findings suggest that PS and GSK-3$\beta $ are required for normal motor protein function, and we propose a model in which PS likely regulates GSK-3$\beta $ activity during transport. These findings have important implications for our understanding of the complex regulatory machinery that must exist \textit{in vivo} and how this system is coordinated during vesicle motility on microtubules. [Preview Abstract] |
Thursday, March 6, 2014 10:12AM - 10:24AM |
S3.00008: Mechanical guidance through cell-cell and cell-surface contact during multicellular streaming Chenlu Wang, Meghan Driscoll, Satyandra K. Gupta, Carole Parent, Wolfgang Losert During collective cell migration, mechanical forces arise from the extracellular matrix (ECM) through cell-surface contact and from other cells through cell-cell contact. These forces regulate the motion of migrating cell groups. To determine how these mechanical interactions balance during cell migration, we measured the shape dynamics of Dictyostelium discoideum cells at the multicellular streaming stage. We found that cells can coordinate their motion by synchronizing protrusion waves that travel along their membranes when they form proper cell-cell adhesion and cell-surface adhesion. In addition, our experiments on live actin labeled cells show that intracellular actin polymerization actively responds to the change of cell-cell/surface adhesion and helps to stabilize multicellular migration streams. Our finding suggests that the coordination of motion between neighboring cells in collective migration requires a balance between cell-cell adhesion and cell-surface adhesion, and that the cell cytoskeleton plays an important role in this balance. [Preview Abstract] |
Thursday, March 6, 2014 10:24AM - 10:36AM |
S3.00009: Model of Exploratory Search for Mating Partners by Fission Yeast Daniel Hurwitz, Felipe Bendezu, Sophie Martin, Dimitrios Vavylonis During conditions of nitrogen starvation, the model eukaryote \textit{S. pombe} (fission yeast) undergoes sexual sporulation. Because fission yeast are non-motile, contact between opposite mating types during spore formation is accomplished by polarizing growth, via the Rho GTP-ase Cdc42, in each mating type towards the selected mate, a process known as shmooing. Recent findings showed that cells pick one of their neighboring compatible mates by randomizing the position of the Cdc42 complex about the cell membrane, such that the complex is stabilized near areas of high concentration of the opposite mating type pheromone. We developed Monte Carlo simulations to model partner finding in populations of mating cells and in small cell clusters. We assume that pheromones are secreted at the site of Cdc42 accumulation and that the Cdc42 dwell time increases in response to increasing pheromone concentration. We measured the number of cells that succeed in successful reciprocal pairing, the number of cells that were unable to find a partner, and the number of cells that picked a partner already engaged with another cell. For optimal cell pairing, we find the pheromone concentration decay length is around 1 micron, of order the cell size. We show that non-linear response of Cdc42 dwell time to pheromone concentration improves the number of successful pairs for a given spatial cell distribution. We discuss how these results compare to non-exploratory pairing mechanisms. [Preview Abstract] |
Thursday, March 6, 2014 10:36AM - 10:48AM |
S3.00010: Centrosomes are autocatalytic droplets of pericentriolar material organized by centrioles David Zwicker, Markus Decker, Steffen Jaensch, Anthony A. Hyman, Frank J\"ulicher We propose a physical description of the centrosome, a membrane-less organelle involved in cell division. In our model, centrosome material occurs in a soluble form in the cytosol and a form that tends to undergo phase separation from the cytosol. We find that an autocatalytic chemical transition between these forms accounts for the temporal evolution observed in experiments. Interestingly, the nucleation of centrosomes can be controlled by an enzymatic activity of the centrioles, which are present at the core of all centrosomes. This non-equilibrium feature also allows for multiple stable centrosomes, a situation which is unstable in equilibrium phase separation. Our theory explains the growth dynamics of centrosomes for all cell sizes down to the eight-cell stage of the \textit{C. elegans} embryo. It also accounts for data acquired in experiments with aberrant numbers of centrosomes and altered cell volumes. Furthermore, our model can describe unequal centrosome sizes observed in cells with disturbed centrioles. Our example suggests a general picture of the organization of membrane-less organelles. [Preview Abstract] |
Thursday, March 6, 2014 10:48AM - 11:00AM |
S3.00011: Quantifying the Dynamic Interactions Between a Clathrin-Coated Pit and Cargo Molecules Aubrey Weigel, Michael Tamkun, Diego Krapf Clathrin-mediated endocytosis is a major pathway of internalization of cargo in eukaryotic cells. This process involves the recruitment of cargo molecules into a growing clathrin-coated pit (CCP). However, cargo-CCP interactions are difficult to study because CCPs display a large degree of lifetime heterogeneity and the interactions with cargo molecules evolve over time. We use single-molecule total internal reflection fluorescence (TIRF) microscopy, in combination with automatic detection and tracking algorithms, to directly visualize the recruitment of individual voltage-gated potassium channels into forming CCPs in living cells. Contrary to widespread ideas, cargo often escapes from a pit before abortive CCP termination or endocytic vesicle production. By measuring tens of thousands of capturing events, we build the distribution of capture times and the times that cargo remains confined to a CCP. An analytical stochastic model is developed and compared to the measured distributions. Due to the dynamic nature of the pit, the model is non-Markovian and it displays long-tail power law statistics. Our findings identify one source of the large heterogeneities observed in CCP maturation and provide a mechanism for the anomalous diffusion of proteins in the plasma membrane. [Preview Abstract] |
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