The “endocytic matrix reloaded” and its impact on the plasticity of migratory strategies

Background and aims: Phenylalanine (Phe) restricted diet, combined with Phe-free L-amino acid supplementation, is the mainstay of treatment for phenylketonuria (PKU). Being the diet a key factor modulating gut microbiota composition, the aim of the present paper was to compare dietary intakes, gut An explosive growth in knowledge, in the last two decades, has conferred a new dimension to the process of endocytosis. Endocytic circuitries have come into focus as a pervasive system that controls virtual all aspects of cell biology. A few years ago, we proposed the term \u201cendocytic matrix\u201d to define a cellular network of signalling wiring that is at the core of the cellular blueprint. A primary role of the endocytic matrix is the delivery of space- and time-resolved signals to the cell in an interpretable format, and, as such, it has profound consequences on polarized cellular and supra-cellular functions, first and foremost, cell motility. Here, we describe a set of recent results that expand this notion and illuminate how endocytic matrix dynamically controls the plasticity of migratory strategies. We further highlight the impact of inter-organelle contact sites on motility and the role of organelle positioning in this process. Finally, we illustrate how global perturbation of the endocytic circuitry influences cellular and supra-cellular mechanics, ultimately controlling a solid-to-liquid-like transition in the mode of motility with potential consequences on cancer dissemination

From filopodia to synapses : the role of actin-capping and anti-capping proteins

Actin-capping and anti-capping proteins are crucial regulators of actin dynamics. Recent studies have indicated that these proteins may be heavily involved in all stages of synaptogenesis, from the emergence of filopodia, through neuritogenesis and synaptic contact stabilization, to the structural changes occurring at the synapse during potentiation phenomena. In this review, we focus on recent evidence pointing to an active role of actin-capping and anti-capping proteins in orchestrating the processes controlling neuronal connectivity and plasticity

Open fracture-dislocation of the knee associated with nonunion of the medial femoral condyle and chronic tendon pa-tellar rupture

Background and aim of work: The incidence of coronal fractures of the femoral condyle, Hoffa frac-tures, ranges from 8.7% to 13% of all fractures of the distal femur and are often observed in polytraumas. Hoffa fractures may be misdiagnosed and consequently not properly treated. Reduction and synthesis of this type of fracture should be achieved to avoid complications such as nonunion, pain, functional impairment. The authors present a case of a 5 year old nonunion of a Hoffa fracture of the medial condyle with chronic patellar tendon rupture. Methods: Revision surgery consisted of reduction and fixation of the Hoffa fracture with screws associated with bone grafting from the iliac crest. Distalization of the patella by Z-plasty and reconstruction of the patellar tendon with Achille’s allograft were also performed. Results: Clinical evaluation after 10 months following the end of the treatment showed a complete resolution of pain, almost complete range of motion, good strength and almost complete functionality of the operated limb. Conclusions: Mistakes in the diagnosis or treatment of Hoffa fracture can often result nonunion, functional impairment, and per-sistent pain. To avoid these, the senior authors of this text believe that the correct treatment of acute Hoffa fracture and its potential associated injuries are crucial, according to the concept of early damage control and later synthesis with soft tissue reconstruction. (www.actabiomedica.it)

Tracking-Free Determination of Single-Cell Displacements and Division Rates in Confluent Monolayers

A biological tissue is an ensemble of soft cells in close physical contact. Events such as cell-shape changes and, more rarely, cell-divisions and apoptosis continuously occur in a tissue, whose collective behavior is set by the cumulative occurrence of such events. In this complex environment, quantifying the single-cell dynamics is key to extract quantitative information to be used to capture the fundamental ingredients of this collective tissue dynamics for validating the predictions of models and numerical simulations. However, tracking the motion of each cell in a dense assembly, even in controlled in vitro settings, is a demanding task, because of a combination of different factors, such as poor image quality, cell shape variability and cell deformability. Here we show that Differential Dynamic Microscopy (DDM), an approach that provides a characterization of the sample structure and dynamics at various spatial frequencies (wave-vectors), can be used successfully to extract quantitative information about a confluent monolayer of Madin-Darby Canine Kidney (MDCK) epithelial cells. In particular, combining structural and dynamical information obtained at different wave-vectors, we show that DDM can provide the single-cell mean squared displacement and the cell division rate at various stages during the temporal evolution of the monolayer. In contrast with tracking algorithms, which require expert supervision and a considerate choice of the analysis parameters, DDM analysis can be run in an automated fashion and yields an unbiased quantification of the dynamic processes under scrutiny, thus providing a powerful means to probe the single-cell dynamics within dense cell collectives

Propagating Cell-Membrane Waves Driven by Curved Activators of Actin Polymerization

Cells exhibit propagating membrane waves which involve the actin cytoskeleton. One type of such membranal waves are Circular Dorsal Ruffles (CDR) which are related to endocytosis and receptor internalization. Experimentally, CDRs have been associated with membrane bound activators of actin polymerization of concave shape. We present experimental evidence for the localization of convex membrane proteins in these structures, and their insensitivity to inhibition of myosin II contractility in immortalized mouse embryo fibroblasts cell cultures. These observations lead us to propose a theoretical model which explains the formation of these waves due to the interplay between complexes that contain activators of actin polymerization and membrane-bound curved proteins of both types of curvature (concave and convex). Our model predicts that the activity of both types of curved proteins is essential for sustaining propagating waves, which are abolished when one type of curved activator is removed. Within this model waves are initiated when the level of actin polymerization induced by the curved activators is higher than some threshold value, which allows the cell to control CDR formation. We demonstrate that the model can explain many features of CDRs, and give several testable predictions. This work demonstrates the importance of curved membrane proteins in organizing the actin cytoskeleton and cell shape

Collagen prolyl hydroxylation-dependent metabolic perturbation governs epigenetic remodeling and mesenchymal transition in pluripotent and cancer cells

Collagen prolyl hydroxylation (CPH), which is catalyzed by prolyl 4-hydroxylase (P4H), is the most prevalent posttranslational modification in humans and requires Vitamin C (VitC). Here we demonstrate that CPH acts as an epigenetic modulator of cell plasticity. Increased CPH induced global DNA/histone methylation in pluripotent stem and tumor cells and promoted cell state transition (CST). Interfering with CPH by either genetic ablation of P4H subunit alpha-2 (P4HA2) or pharmacologic treatment reverted epigenetic changes and antagonized CST. Mechanistically, we suggest that CPH modifies the epigenetic landscape by reducing VitC for DNA and histone demethylases. Repurposed drugs targeting CPH-mediated metabolic perturbation, such as the antiasthmatic Budesonide, blocked metastatic dissemination of breast cancer cells in vivo by preventing mesenchymal transition. Our study provides mechanistic insights into how metabolic cues and epigenetic factors integrate to control cell state transition and paves the way for the development of novel antimetastatic strategies. Significance: A phenotype-based high-throughput screening reveals unforeseen metabolic control of cell plasticity and identifies budesonide as a drug candidate for metastatic cancer

IRSp53 controls plasma membrane shape and polarized transport at the nascent lumen in epithelial tubules

It is unclear whether the establishment of apical–basal cell polarity during the generation of epithelial lumens requires molecules acting at the plasma membrane/actin interface. Here, we show that the I-BAR-containing IRSp53 protein controls lumen formation and the positioning of the polarity determinants aPKC and podocalyxin. Molecularly, IRSp53 acts by regulating the localization and activity of the small GTPase RAB35, and by interacting with the actin capping protein EPS8. Using correlative light and electron microscopy, we further show that IRSp53 ensures the shape and continuity of the opposing plasma membrane of two daughter cells, leading to the formation of a single apical lumen. Genetic removal of IRSp53 results in abnormal renal tubulogenesis, with altered tubular polarity and architectural organization. Thus, IRSp53 acts as a membrane curvature-sensing platform for the assembly of multi-protein complexes that control the trafficking of apical determinants and the integrity of the luminal plasma membrane

IRSp53 controls plasma membrane shape and polarized transport at the nascent lumen in epithelial tubules

It is unclear whether the establishment of apical\u2013basal cell polarity during the generation of epithelial lumens requires molecules acting at the plasma membrane/actin interface. Here, we show that the I-BAR-containing IRSp53 protein controls lumen formation and the positioning of the polarity determinants aPKC and podocalyxin. Molecularly, IRSp53 acts by regulating the localization and activity of the small GTPase RAB35, and by interacting with the actin capping protein EPS8. Using correlative light and electron microscopy, we further show that IRSp53 ensures the shape and continuity of the opposing plasma membrane of two daughter cells, leading to the formation of a single apical lumen. Genetic removal of IRSp53 results in abnormal renal tubulogenesis, with altered tubular polarity and architectural organization. Thus, IRSp53 acts as a membrane curvature-sensing platform for the assembly of multi-protein complexes that control the trafficking of apical determinants and the integrity of the luminal plasma membrane

LIN7 regulates the filopodia and neurite promoting activity of IRSp53

The insulin receptor substrate protein of 53\u2005kDa (IRSp53) is critically involved in the formation of filopodia and neurites through mechanisms that have only in part been clarified. Here, we investigated the role of the small scaffold protein LIN7, an interactor of IRSp53. We found that formation of actin-filled protrusions in neuronal NSC34 cells and neurites in neuroblastoma N2A depends on motifs mediating the LIN7:IRSp53 association, as both the coexpression of LIN7 with IRSp53 or the expression of the L27-IRSp53 chimera (a fusion protein between IRSp53 and the LIN7L27 domain for plasma membrane protein complexes association) prevented actin-deficient protrusions induced by overexpressed IRSp53, and enhanced the formation of actin-filled protrusions. The regulatory role of LIN7 in IRSp53-mediated extension of filopodia was demonstrated by live-cell imaging experiments in neuronal N2A cells. Moreover, LIN7 silencing prevented the extension of filopodia and neurites, induced by ectopic expression of IRSp53 or serum starvation, respectively in undifferentiated and differentiated N2A cells. The expression of full length IRSp53 or the LIN7\u394PDZ mutant lacking the domain for association with IRSp53 was unable to restore neuritogenesis in LIN7 silenced cells. Conversely, defective neuritogenesis could be rescued by the expression of RNAi-resistant full length LIN7 or chimeric L27-IRSp53. Finally, LIN7 silencing prevented the recruitment of IRSp53 in Triton X-100 insoluble complexes, otherwise occurring in differentiated cells. Collectively these data indicate that LIN7 is a novel regulator of IRSp53, and that their association is required to promote the formation of actin-dependent filopodia and neurites

The GTPase-activating protein RN-tre controls focal adhesion turnover and cell migration.

SummaryBackgroundIntegrin-mediated adhesion of cells to the extracellular matrix (ECM) relies on the dynamic formation of focal adhesions (FAs), which are biochemical and mechanosensitive platforms composed of a large variety of cytosolic and transmembrane proteins. During migration, there is a constant turnover of ECM contacts that initially form as nascent adhesions at the leading edge, mature into FAs as actomyosin tension builds up, and are then disassembled at the cell rear, thus allowing for cell detachment. Although the mechanisms of FA assembly have largely been defined, the molecular circuitry that regulates their disassembly still remains elusive.ResultsHere, we show that RN-tre, a GTPase-activating protein (GAP) for Rabs including Rab5 and Rab43, is a novel regulator of FA dynamics and cell migration. RN-tre localizes to FAs and to a pool of Rab5-positive vesicles mainly associated with FAs undergoing rapid remodeling. We found that RN-tre inhibits endocytosis of β1, but not β3, integrins and delays the turnover of FAs, ultimately impairing β1-dependent, but not β3-dependent, chemotactic cell migration. All of these effects are mediated by its GAP activity and rely on Rab5.ConclusionsOur findings identify RN-tre as the Rab5-GAP that spatiotemporally controls FA remodeling during chemotactic cell migration