Actomyosin-dependent dynamic spatial patterns of cytoskeletal components drive mesoscale podosome organization

Podosomes are cytoskeletal structures crucial for cell protrusion and matrix remodelling in osteoclasts, activated endothelial cells, macrophages and dendritic cells. In these cells, hundreds of podosomes are spatially organized in diversely shaped clusters. Although we and others established individual podosomes as micron-sized mechanosensing protrusive units, the exact scope and spatiotemporal organization of podosome clustering remain elusive. By integrating a newly developed extension of Spatiotemporal Image Correlation Spectroscopy with novel image analysis, we demonstrate that F-actin, vinculin and talin exhibit directional and correlated flow patterns throughout podosome clusters. Pattern formation and magnitude depend on the cluster actomyosin machinery. Indeed, nanoscopy reveals myosin IIA-decorated actin filaments interconnecting multiple proximal podosomes. Extending well-beyond podosome nearest neighbours, the actomyosin-dependent dynamic spatial patterns reveal a previously unappreciated mesoscale connectivity throughout the podosome clusters. This directional transport and continuous redistribution of podosome components provides a mechanistic explanation of how podosome clusters function as coordinated mechanosensory area

Modular actin nano-architecture enables podosome protrusion and mechanosensing

Basement membrane transmigration during embryonal development, tissue homeostasis and tumor invasion relies on invadosomes, a collective term for invadopodia and podosomes. An adequate structural framework for this process is still missing. Here, we reveal the modular actin nano-architecture that enables podosome protrusion and mechanosensing. The podosome protrusive core contains a central branched actin module encased by a linear actin module, each harboring specific actin interactors and actin isoforms. From the core, two actin modules radiate: ventral filaments bound by vinculin and connected to the plasma membrane and dorsal interpodosomal filaments crosslinked by myosin IIA. On stiff substrates, the actin modules mediate long-range substrate exploration, associated with degradative behavior. On compliant substrates, the vinculin-bound ventral actin filaments shorten, resulting in short-range connectivity and a focally protrusive, non-degradative state. Our findings redefine podosome nanoscale architecture and reveal a paradigm for how actin modularity drives invadosome mechanosensing in cells that breach tissue boundaries

Application of Rapid Fluorescence Lifetime Imaging Microscopy (RapidFLIM) to Examine Dynamics of Nanoparticle Uptake in Live Cells

A key challenge in nanomedicine stems from the continued need for a systematic understanding of the delivery of nanoparticles in live cells. Complexities in delivery are often influenced by the biophysical characteristics of nanoparticles, where even subtle changes to nanoparticle designs can alter cellular uptake, transport and activity. Close examination of these processes, especially with imaging, offers important insights that can aid in future nanoparticle design or translation. Rapid fluorescence lifetime imaging microscopy (RapidFLIM) is a potentially valuable technology for examining intracellular mechanisms of nanoparticle delivery by directly correlating visual data with changes in the biological environment. To date, applications for this technology in nanoparticle research have not been explored. A PicoQuant RapidFLIM system was used together with commercial silica nanoparticles to follow particle uptake in glioblastoma cells. Importantly, RapidFLIM imaging showed significantly improved image acquisition speeds over traditional FLIM, which enabled the tracking of nanoparticle uptake into subcellular compartments. We determined mean lifetime changes and used this to delineate significant changes in nanoparticle lifetimes (>0.39 ns), which showed clustering of these tracks proximal to both extracellular and nuclear membrane boundaries. These findings demonstrate the ability of RapidFLIM to track, localize and quantify changes in single nanoparticle fluorescence lifetimes and highlight RapidFLIM as a valuable tool for multiparameter visualization and analysis of nanoparticle molecular dynamics in live cells

Impact of Expressive Wrinkles on Perception of a Virtual Character’s Facial Expressions of Emotions

Facial animation has reached a high level of photorealism. Skin is rendered with grain and translucency, wrinkles are accurate and dynamic. These recent visual improvements are not fully tested for their contribution to the perceived expressiveness of virtual characters. This paper presents a perceptual study assessing the impact of different rendering modes of expressive wrinkles on users’ perception of facial expressions of basic and complex emotions. Our results suggest that realistic wrinkles increase agent’s expressivity and user’s preference, but not the recognition of emotion categories. This study was conducted using our real time facial animation platform that is designed for perceptive evaluations of affective interaction

Autonomous actors in networked collaborative virtual environments

Introducing seemingly autonomous virtual beings into virtual environments to co-habit and collaborate with us is a continuous challenge and source of interest. Latest proof of human excitement for virtual life is the current worldwide craze for electronic pets that must be fed and cared for lest they develop a bad character or die. Even more interesting is the inclusion of autonomous actors in networked collaborative virtual environments (NCVEs). They provide a meeting place for people from different geographical locations and virtual beings. In NCVEs one doesn't see correspondents, only their graphical representations in the virtual world, which is the same as for the virtual ones-therefore the communication with virtual beings can come naturally. There is no single solution to the simulation of autonomous behavior. This is an ongoing research topic. Therefore it is interesting to provide an open NCVE system for easy interfacing with various implementations of autonomous behavior. In this way, the system can serve as an application platform with existing algorithms, as well as a research testbed for new autonomous behavior algorithms. The paper studies the requirements for such an open interface and, based on this study, presents an implementation within the Virtual Life Network (VLNET) system. Results are presented in terms of two case studies. A simple one implementing a dumb servant character and a more complex one connecting VLNET with the autonomous agent program Eliza (Weizenbaum, 1966

A flexible architecture for virtual humans in networked collaborative virtual environments

Complex virtual human representation provides more natural interaction and communication among participants in networked virtual environments, hence it is expected to increase the sense of being together within the same virtual world. We present a flexible framework for the integration of virtual humans in networked collaborative virtual environments. A modular architecture allows flexible representation and control of the virtual humans, whether they are controlled by a physical user using all sorts of tracking and other devices, or by an intelligent control program turning them into autonomous actors. The modularity of the system allows for fairly easy extensions and integration with new techniques making it interesting also as a testbed for various domains from “classic” VR to psychological experiments. We present results in terms of functionalities, example applications and measurements of performance and network traffic with an increasing number of participants in the simulatio