a-graphical-user-interface-for-software-assisted-tracking-protein

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

University of Muenster

The insulin/IGF-1 signaling pathway controls cellular and organismal growth in many multicellular organisms. In Drosophila, genetic defects in components of the insulin signaling pathway produce small flies that are delayed in development and possess fewer and smaller cells as well as female sterility, reminiscent of the phenotypes of starved flies.

Nutrient-dependent expression of insulin-like peptides from neuroendocrine cells in the CNS contributes to growth regulation in Drosophila.

Insulin and insulin-like growth factors (IGFs) signal through a highly conserved pathway and control growth and metabolism in both vertebrates and invertebrates. In mammals, insulin-like growth factor binding proteins (IGFBPs) bind IGFs with high affinity and modulate their mitogenic, anti-apoptotic and metabolic actions, but no functional homologs have been identified in invertebrates so far.

Imp-L2, a putative homolog of vertebrate IGF-binding protein 7, counteracts insulin signaling in Drosophila and is essential for starvation resistance.

Polarization of tissue is achieved by asymmetric distribution of proteins and organelles within individual cells. However, existing quantitative assays to measure this asymmetry in an automated and unbiased manner suffer from significant limitations.

A universal analysis tool for the detection of asymmetric signal distribution in microscopic images.

Many of the more than 20 mammalian proteins with N-BAR domains control cell architecture and endocytosis by associating with curved sections of the plasma membrane. It is not well understood whether N-BAR proteins are recruited directly by processes that mechanically curve the plasma membrane or indirectly by plasma-membrane-associated adaptor proteins that recruit proteins with N-BAR domains that then induce membrane curvature. Here, we show that externally induced inward deformation of the plasma membrane by cone-shaped nanostructures (nanocones) and internally induced inward deformation by contracting actin cables both trigger recruitment of isolated N-BAR domains to the curved plasma membrane. Markedly, live-cell imaging in adherent cells showed selective recruitment of full-length N-BAR proteins and isolated N-BAR domains to plasma membrane sub-regions above nanocone stripes. Electron microscopy confirmed that N-BAR domains are recruited to local membrane sites curved by nanocones. We further showed that N-BAR domains are periodically recruited to curved plasma membrane sites during local lamellipodia retraction in the front of migrating cells. Recruitment required myosin-II-generated force applied to plasma-membrane-connected actin cables. Together, our results show that N-BAR domains can be directly recruited to the plasma membrane by external push or internal pull forces that locally curve the plasma membrane.

External push and internal pull forces recruit curvature-sensing N-BAR domain proteins to the plasma membrane.

Vesicle endocytosis at the plasma membrane is associated with a precise temporal choreography in the recruitment of cytosolic proteins that sense, generate, or stabilize locally curved membrane regions. To dissect the role of membrane curvature sensing from other co-occurring events during the initial steps of endocytosis, we developed a method to artificially induce nanoscale deformations of the PM in living cells that is based on cone-shaped nanostructures (i.e., Nanocones). When cultured on Nanocones, cells create stable inward plasma membrane deformations to which curvature-sensing proteins are recruited. Here, we provide a detailed protocol how to use Nanocones to study recruitment during the initial steps of endocytosis in cells by fluorescence and electron microscopy.

Nanocones to study initial steps of endocytosis.

In the vertebrate central nervous system, exploratory filopodia transiently form on dendritic branches to sample the neuronal environment and initiate new trans-neuronal contacts. While much is known about the molecules that control filopodia extension and subsequent maturation into functional synapses, the mechanisms that regulate initiation of these dynamic, actin-rich structures have remained elusive. Here, we find that filopodia initiation is suppressed by recruitment of ArhGAP44 to actin-patches that seed filopodia. Recruitment is mediated by binding of a membrane curvature-sensing ArhGAP44 N-BAR domain to plasma membrane sections that were deformed inward by acto-myosin mediated contractile forces. A GAP domain in ArhGAP44 triggers local Rac-GTP hydrolysis, thus reducing actin polymerization required for filopodia formation. Additionally, ArhGAP44 expression increases during neuronal development, concurrent with a decrease in the rate of filopodia formation. Together, our data reveals a local auto-regulatory mechanism that limits initiation of filopodia via protein recruitment to nanoscale membrane deformations.

Dynamic recruitment of the curvature-sensitive protein ArhGAP44 to nanoscale membrane deformations limits exploratory filopodia initiation in neurons.

Force-regulation at cellular membranes relies on dynamic molecular platforms that integrate intra- and extracellular signals to control cell shape and function. To correctly respond to a continuously changing environment, activity of these platforms needs to be tightly controlled in space and time. Over the last few years, curvature-dependent mechano-chemical signal translation—a receptor-independent signaling mechanism where physical forces at the plasma membrane trigger nanoscale membrane deformations that are then translated into chemical signal transduction cascades—has emerged as a new signaling principle that cells use to regulate forces at the membrane. However, until recently, technical limitations have precluded studies of this force-induced curvature-dependent signaling at the physiological scale. Here, we comment on recent advancements that allow studying curvature-dependent signaling at membranes, and discuss processes where it may be involved in. Considering its general impact on cell function, a particular focus will be put on the curvature-dependence of feedback loops that control actin-based forces at cellular membranes.

Force-control at cellular membranes.

Planar cell polarity, the polarization of cells within the plane of the epithelium, orthogonal to the apical-basal axis, is essential for a growing list of developmental events, and - over the last 15 years - has evolved from a little-studied curiosity in Drosophila to the subject of a substantial research enterprise. In that time, it has been recognized that two molecular systems are responsible for polarization of most tissues: Both the "core" Frizzled system and the "global" Fat/Dachsous/Four-jointed system produce molecular asymmetry within cells, and contribute to morphological polarization. In this review, we discuss recent findings on the molecular mechanism that links "global" directional signals with local coordinated polarity.

Polarized trafficking provides spatial cues for planar cell polarization within a tissue.

Studies of cellular surface features gain from correlative approaches, where live cell information acquired by fluorescence light microscopy is complemented by ultrastructural information from scanning electron micrographs. Current approaches to spatially align fluorescence images with scanning electron micrographs are technically challenging and often cost or time-intensive. Relying exclusively on open-source software and equipment available in a standard lab, we have developed a method for rapid, software-assisted alignment of fluorescence images with the corresponding scanning electron micrographs via a stochastic gold micro-pattern. Here, we provide detailed instructions for micro-pattern production and image processing, troubleshooting for critical intermediate steps, and examples of membrane ultra-structures aligned with the fluorescence signal of proteins enriched at such sites. Together, the presented method for correlative fluorescence - scanning electron microscopy is versatile, robust and easily integrated into existing workflows, permitting image alignment with accuracy comparable to existing approaches with negligible investment of time or capital.

Stochastic Micro-Pattern for Automated Correlative Fluorescence - Scanning Electron Microscopy.

Hippocampal long-term potentiation (LTP) represents the cellular response of excitatory synapses to specific patterns of high neuronal activity and is required for learning and memory. Here we identify a mechanism that requires the calcium-binding protein Copine-6 to translate the initial calcium signals into changes in spine structure. We show that Copine-6 is recruited from the cytosol of dendrites to postsynaptic spine membranes by calcium transients that precede LTP. Cpne6 knockout mice are deficient in hippocampal LTP, learning and memory. Hippocampal neurons from Cpne6 knockouts lack spine structural plasticity as do wild-type neurons that express a Copine-6 calcium mutant. The function of Copine-6 is based on its binding, activating and recruiting the Rho GTPase Rac1 to cell membranes. Consistent with this function, the LTP deficit of Cpne6 knockout mice is rescued by the actin stabilizer jasplakinolide. These data show that Copine-6 links activity-triggered calcium signals to spine structural plasticity necessary for learning and memory.

The calcium sensor Copine-6 regulates spine structural plasticity and learning and memory.

Filopodia are dynamic, actin-rich structures that transiently form on a variety of cell types. To understand the underlying control mechanisms requires precise monitoring of localization and concentration of individual regulatory and structural proteins as filopodia elongate and subsequently retract. Although several methods exist that analyze changes in filopodial shape, a software solution to reliably correlate growth dynamics with spatially resolved protein concentration along the filopodium independent of bending, lateral shift, or tilting is missing. Here we introduce a novel approach based on the convex-hull algorithm for parallel analysis of growth dynamics and relative spatiotemporal protein concentration along flexible filopodial protrusions. Detailed in silico tests using various geometries confirm that our technique accurately tracks growth dynamics and relative protein concentration along the filopodial length for a broad range of signal distributions. To validate our technique in living cells, we measure filopodial dynamics and quantify spatiotemporal localization of filopodia-associated proteins during the filopodial extension-retraction cycle in a variety of cell types in vitro and in vivo. Together these results show that the technique is suitable for simultaneous analysis of growth dynamics and spatiotemporal protein enrichment along filopodia. To allow readily application by other laboratories, we share source code and instructions for software handling.

Automated analysis of filopodial length and spatially resolved protein concentration via adaptive shape tracking.

Many core paradigms of contemporary neuroscience are based on information obtained by electron or light microscopy. Intriguingly, these two imaging techniques are often viewed as complementary, yet separate entities. Recent technological advancements in microscopy techniques, labeling tools, and fixation or preparation procedures have fueled the development of a series of hybrid approaches that allow correlating functional fluorescence microscopy data and ultrastructural information from electron micrographs from a singular biological event. As correlative light electron microscopy (CLEM) approaches become increasingly accessible, long-standing neurobiological questions regarding structure-function relation are being revisited. In this review, we will survey what developments in electron and light microscopy have spurred the advent of correlative approaches, highlight the most relevant CLEM techniques that are currently available, and discuss its potential and limitations with respect to neuronal and synapse-specific applications.

Milos Galic

DFG Cluster of Excellence 'Cells in Motion',
(EXC 1003),
Institute of Medical Physics and Biophysics

University of Muenster

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Milos Galic

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}DFG Cluster of Excellence 'Cells in Motion',(EXC 1003),Institute of Medical Physics and Biophysics

University of Muenster

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

DFG Cluster of Excellence 'Cells in Motion',
(EXC 1003),
Institute of Medical Physics and Biophysics