Simultaneous quantification of actin monomer and filament dynamics with modelling assisted analysis of photoactivation

Photoactivation allows one to pulse-label molecules and obtain quantitative data about their behavior. We have devised a new modeling-based analysis for photoactivatable actin experiments that simultaneously measures properties of monomeric and filamentous actin in a three-dimensional cellular environment. We use this method to determine differences in the dynamic behavior of β- and γ-actin isoforms, showing that both inhabit filaments that depolymerize at equal rates but that β-actin exists in a higher monomer-to-filament ratio. We also demonstrate that cofilin (cofilin 1) equally accelerates depolymerization of filaments made from both isoforms, but is only required to maintain the β-actin monomer pool. Finally, we used modeling-based analysis to assess actin dynamics in axon-like projections of differentiating neuroblastoma cells, showing that the actin monomer concentration is significantly depleted as the axon develops. Importantly, these results would not have been obtained using traditional half-time analysis. Given that parameters of the publicly available modeling platform can be adjusted to suit the experimental system of the user, this method can easily be used to quantify actin dynamics in many different cell types and subcellular compartments

In Silico Generation of Alternative Hypotheses Using Causal Mapping (CMAP)

Previously, we introduced causal mapping (CMAP) as an easy to use systems biology tool for studying the behavior of biological processes that occur at the cellular and molecular level. CMAP is a coarse-grained graphical modeling approach in which the system of interest is modeled as an interaction map between functional elements of the system, in a manner similar to portrayals of signaling pathways commonly used by molecular cell biologists. CMAP describes details of the interactions while maintaining the simplicity of other qualitative methods (e.g., Boolean networks)

Compression and dilation of the membrane-cortex layer generates rapid changes in cell shape

A cyclic process of membrane-cortex compression and dilation generates a traveling wave of cortical actin density that in turn generates oscillations in cell morphology.Rapid changes in cellular morphology require a cell body that is highly flexible yet retains sufficient strength to maintain structural integrity. We present a mechanism that meets both of these requirements. We demonstrate that compression (folding) and subsequent dilation (unfolding) of the coupled plasma membrane–cortex layer generates rapid shape transformations in rounded cells. Two- and three-dimensional live-cell images showed that the cyclic process of membrane-cortex compression and dilation resulted in a traveling wave of cortical actin density. We also demonstrate that the membrane-cortex traveling wave led to amoeboid-like cell migration. The compression–dilation hypothesis offers a mechanism for large-scale cell shape transformations that is complementary to blebbing, where the plasma membrane detaches from the actin cortex and is initially unsupported when the bleb extends as a result of cytosolic pressure. Our findings provide insight into the mechanisms that drive the rapid morphological changes that occur in many physiological contexts, such as amoeboid migration and cytokinesis

Spatiotemporal relationships between the cell shape and the actomyosin cortex of periodically protruding cells

We investigate the dynamics of cell shape and analyze the actin and myosin distributions of cells exhibiting cortical density traveling waves. These waves propagate by repeated cycles of cortical compression (folding) and dilation (unfolding) that lead to periodic protrusions (oscillations) of the cell boundary. The focus of our detailed analysis is the remarkable periodicity of this phenotype, in which both the overall shape transformation and distribution of actomyosin density are repeated from cycle to cycle even though the characteristics of the shape transformation vary significantly for different regions of the cell. We show, using correlation analysis, that during traveling wave propagation cortical actin and plasma membrane densities are tightly coupled at each point along the cell periphery. We also demonstrate that the major protrusion appears at the wave trailing edge just after the actin cortex density has reached a maximum. Making use of the extraordinary periodicity, we employ latrunculin to demonstrate that sequestering actin monomers can have two distinct effects: low latrunculin concentrations can trigger and enhance traveling waves but higher concentrations of this drug retard the waves. The fundamental mechanism underlying this periodically protruding phenotype, involving folding and unfolding of the cortex-membrane couple, is likely to hold important clues for diverse phenomena including cell division and amoeboid-type migration

A Conformational Transition State Accompanies Tryptophan Activation by B. stearothermophilus Tryptophanyl-tRNA Synthetase

B. stearothermophilus tryptophanyl-tRNA synthetase catalysis proceeds via high-energy protein conformations. Unliganded MD trajectories of the Pre-Transition-state complex with Mg2+•ATP and the (post) transition-state analog complex with adenosine tetraphosphate relax rapidly in opposite directions, the former regressing, the latter progressing along the structural reaction coordinate. The two crystal structures (RMSD 0.7 Å) therefore lie on opposite sides of a conformational free energy maximum as the chemical transition state forms. SNAPP analysis illustrates the complexity of the associated long-range conformational coupling. Switching interactions in four non-polar core regions are locally isoenergetic throughout the transition. Different configurations, however, propagate their effects to unfavorable, longer-range interactions at the molecular surface. Designed mutation shows that switching interactions enhance the rate, perhaps by destabilizing the ground state immediately before the transition state and limiting non-productive diffusion before and after the chemical transition state, thereby reducing the activation entropy. This paradigm may apply broadly to energy-transducing enzymes

Modeling capping protein FRAP and CALI experiments reveals in vivo regulation of actin dynamics

To gain insights on cellular mechanisms regulating actin polymerization, we used the Virtual Cell to model FRAP and chromophore assisted laser inactivation (CALI) experiments on EGFP-capping protein (EGFP-CP). Modeling the FRAP kinetics demonstrated that the in vivo rate for the dissociation of CP from actin filaments is much faster (~0.1 s−1) than that measured in vitro (0.01–0.0004 s−1). The CALI simulation revealed that in order to induce sustainable changes in cell morphology after CP inactivation, the cells should exhibit anti-capping ability. We included the VASP protein as the anti-capping agent in the modeling scheme. The model predicts that VASP affinity for barbed ends has a cooperative dependence on the concentration of VASP-barbed end complexes. This dependence produces a positive feedback that stabilizes the complexes and allows sustained growth at clustered filament tips. We analyzed the range of laser intensities that are sufficient to induce changes in cell morphology. This analysis demonstrates that FRAP experiments with EGFP-CP can be performed safely without changes in cell morphology, because, the intensity of the photobleaching beam is not high enough to produce the critical concentration of free barbed ends that will induce filament growth before diffusional replacement of EGFP-CP occurs

Modeling the Excess Cell Surface Stored in a Complex Morphology of Bleb-Like Protrusions

Cells transition from spread to rounded morphologies in diverse physiological contexts including mitosis and mesenchymal-to-amoeboid transitions. When these drastic shape changes occur rapidly, cell volume and surface area are approximately conserved. Consequently, the rounded cells are suddenly presented with a several-fold excess of cell surface whose area far exceeds that of a smooth sphere enclosing the cell volume. This excess is stored in a population of bleb-like protrusions (BLiPs), whose size distribution is shown by electron micrographs to be skewed. We introduce three complementary models of rounded cell morphologies with a prescribed excess surface area. A 2D Hamiltonian model provides a mechanistic description of how discrete attachment points between the cell surface and cortex together with surface bending energy can generate a morphology that satisfies a prescribed excess area and BLiP number density. A 3D random seed-and-growth model simulates efficient packing of BLiPs over a primary rounded shape, demonstrating a pathway for skewed BLiP size distributions that recapitulate 3D morphologies. Finally, a phase field model (2D and 3D) posits energy-based constitutive laws for the cell membrane, nematic F-actin cortex, interior cytosol, and external aqueous medium. The cell surface is equipped with a spontaneous curvature function, a proxy for the cell surface-cortex couple, that is a priori unknown, which the model “learns” from the thin section transmission electron micrograph image (2D) or the “seed and growth” model image (3D). Converged phase field simulations predict self-consistent amplitudes and spatial localization of pressure and stress throughout the cell for any posited stationary morphology target and cell compartment constitutive properties. The models form a general framework for future studies of cell morphological dynamics in a variety of biological contexts

Nonlytic cellular release of hepatitis A virus requires dual capsid recruitment of the ESCRT-associated Bro1 domain proteins HD-PTP and ALIX

Although picornaviruses are conventionally considered ‘nonenveloped’, members of multiple picornaviral genera are released nonlytically from infected cells in extracellular vesicles. The mechanisms underlying this process are poorly understood. Here, we describe interactions of the hepatitis A virus (HAV) capsid with components of host endosomal sorting complexes required for transport (ESCRT) that play an essential role in release. We show release of quasi-enveloped virus (eHAV) in exosome-like vesicles requires a conserved export signal located within the 8 kDa C-terminal VP1 pX extension that functions in a manner analogous to late domains of canonical enveloped viruses. Fusing pX to a self-assembling engineered protein nanocage (EPN-pX) resulted in its ESCRT-dependent release in extracellular vesicles. Mutational analysis identified a 24 amino acid peptide sequence located within the center of pX that was both necessary and sufficient for nanocage release. Deleting a YxxL motif within this sequence ablated eHAV release, resulting in virus accumulating intracellularly. The pX export signal is conserved in non-human hepatoviruses from a wide range of mammalian species, and functional in pX sequences from bat hepatoviruses when fused to the nanocage protein, suggesting these viruses are released as quasi-enveloped virions. Quantitative proteomics identified multiple ESCRT-related proteins associating with EPN-pX, including ALG2-interacting protein X (ALIX), and its paralog, tyrosine-protein phosphatase non-receptor type 23 (HD-PTP), a second Bro1 domain protein linked to sorting of ubiquitylated cargo into multivesicular endosomes. RNAi-mediated depletion of either Bro1 domain protein impeded eHAV release. Super-resolution fluorescence microscopy demonstrated colocalization of viral capsids with endogenous ALIX and HD-PTP. Co-immunoprecipitation assays using biotin-tagged peptides and recombinant proteins revealed pX interacts directly through the export signal with N-terminal Bro1 domains of both HD-PTP and ALIX. Our study identifies an exceptionally potent viral export signal mediating extracellular release of virus-sized protein assemblies and shows release requires non-redundant activities of both HD-PTP and ALIX

Two Functionally Distinct Sources of Actin Monomers Supply the Leading Edge of Lamellipodia

Lamellipodia, the sheet-like protrusions of motile cells, consist of networks of actin filaments (F-actin) regulated by the ordered assembly from and disassembly into actin monomers (G-actin). Traditionally, G-actin is thought to exist as a homogeneous pool. Here, we show that there are two functionally and molecularly distinct sources of G-actin that supply lamellipodial actin networks. G-actin originating from the cytosolic pool requires the monomer-binding protein thymosin β4 (Tβ4) for optimal leading-edge localization, is targeted to formins, and is responsible for creating an elevated G/F-actin ratio that promotes membrane protrusion. The second source of G-actin comes from recycled lamellipodia F-actin. Recycling occurs independently of Tβ4 and appears to regulate lamellipodia homeostasis. Tβ4-bound G-actin specifically localizes to the leading edge because it does not interact with Arp2/3-mediated polymerization sites found throughout the lamellipodia. These findings demonstrate that actin networks can be constructed from multiple sources of monomers with discrete spatiotemporal functions