Invadopodia Methods: Detection of Invadopodia Formation and Activity in Cancer Cells Using Reconstituted 2D and 3D Collagen-Based Matrices.

Tumor dissemination involves cancer cell migration through the extracellular matrix (ECM). ECM is mainly composed of collagen fibers that oppose cell invasion. To overcome hindrance in the matrix, cancer cells deploy a protease-dependent program in order to remodel the matrix fibers. Matrix remodeling requires the formation of actin-based matrix/plasma membrane contact sites called invadopodia, responsible for collagen cleavage through the accumulation and activity of the transmembrane type-I matrix metalloproteinase (MT1-MMP). In this article, we describe experimental procedures designed to assay for invadopodia formation and for invadopodia activity using 2D and 3D models based on gelatin (denatured collagen) and fibrillar type-I collagen matrices.

A mechanosensitive caveolae-invadosome interplay drives matrix remodelling for cancer cell invasion.

Invadosomes and caveolae are mechanosensitive structures that are implicated in metastasis. Here, we describe a unique juxtaposition of caveola clusters and matrix degradative invadosomes at contact sites between the plasma membrane of cancer cells and constricting fibrils both in 2D and 3D type I collagen matrix environments. Preferential association between caveolae and straight segments of the fibrils, and between invadosomes and bent segments of the fibrils, was observed along with matrix remodelling. Caveola recruitment precedes and is required for invadosome formation and activity. Reciprocally, invadosome disruption results in the accumulation of fibril-associated caveolae. Moreover, caveolae and the collagen receptor ß1 integrin co-localize at contact sites with the fibrils, and integrins control caveola recruitment to fibrils. In turn, caveolae mediate the clearance of ß1 integrin and collagen uptake in an invadosome-dependent and collagen-cleavage-dependent mechanism. Our data reveal a reciprocal interplay between caveolae and invadosomes that coordinates adhesion to and proteolytic remodelling of confining fibrils to support tumour cell dissemination.

Codependencies of mTORC1 signaling and endolysosomal actin structures.

The mechanistic target of rapamycin complex 1 (mTORC1) is part of the amino acid sensing machinery that becomes activated on the endolysosomal surface in response to nutrient cues. Branched actin generated by WASH and Arp2/3 complexes defines endolysosomal microdomains. Here, we find mTORC1 components in close proximity to endolysosomal actin microdomains. We investigated for interactors of the mTORC1 lysosomal tether, RAGC, by proteomics and identified multiple actin filament capping proteins and their modulators. Perturbation of RAGC function affected the size of endolysosomal actin, consistent with a regulation of actin filament capping by RAGC. Reciprocally, the pharmacological inhibition of actin polymerization or alteration of endolysosomal actin obtained upon silencing of WASH or Arp2/3 complexes impaired mTORC1 activity. Mechanistically, we show that actin is required for proper association of RAGC and mTOR with endolysosomes. This study reveals an unprecedented interplay between actin and mTORC1 signaling on the endolysosomal system.

mTOR Repression in Response to Amino Acid Starvation Promotes ECM Degradation Through MT1-MMP Endocytosis Arrest.

Under conditions of starvation, normal and tumor epithelial cells can rewire their metabolism toward the consumption of extracellular proteins, including extracellular matrix-derived components as nutrient sources. The mechanism of pericellular matrix degradation by starved cells has been largely overlooked. Here it is shown that matrix degradation by breast and pancreatic tumor cells and patient-derived xenograft explants increases by one order of magnitude upon amino acid and growth factor deprivation. In addition, it is found that collagenolysis requires the invadopodia components, TKS5, and the transmembrane metalloproteinase, MT1-MMP, which are key to the tumor invasion program. Increased collagenolysis is controlled by mTOR repression upon nutrient depletion or pharmacological inhibition by rapamycin. The results reveal that starvation hampers clathrin-mediated endocytosis, resulting in MT1-MMP accumulation in arrested clathrin-coated pits. The study uncovers a new mechanism whereby mTOR repression in starved cells leads to the repurposing of abundant plasma membrane clathrin-coated pits into robust ECM-degradative assemblies.

A Portable Passive Rehabilitation Robot for Upper-Extremity Functional Resistance Training.

OBJECTIVE: Loss of arm function is common in individuals with neurological damage, such as stroke or cerebral palsy. Robotic devices that address muscle strength deficits in a task-specific manner can assist in the recovery of arm function; however, current devices are typically large, bulky, and expensive to be routinely used in the clinic or at home. This study sought to address this issue by developing a portable planar passive rehabilitation robot, PaRRo. METHODS: We designed PaRRo with a mechanical layout that incorporated kinematic redundancies to generate forces that directly oppose the user's movement. Cost-efficient eddy current brakes were used to provide scalable resistances. The lengths of the robot's linkages were optimized to have a reasonably large workspace for human planar reaching. We then performed theoretical analysis of the robot's resistive force generating capacity and steerable workspace using MATLAB simulations. We also validated the device by having a subject move the end-effector along different paths at a set velocity using a metronome while simultaneously collecting surface electromyography (EMG) and end-effector forces felt by the user. RESULTS: Results from simulation experiments indicated that the robot was capable of producing sufficient end-effector forces for functional resistance training. We also found the endpoint forces from the user were similar to the theoretical forces expected at any direction of motion. EMG results indicated that the device was capable of providing adjustable resistances based on subjects' ability levels, as the muscle activation levels scaled with increasing magnet exposures. CONCLUSION: These results indicate that PaRRo is a feasible approach to provide functional resistance training to the muscles along the upper extremity. SIGNIFICANCE: The proposed robotic device could provide a technological breakthrough that will make rehabilitation robots accessible for small outpatient rehabilitation centers and in-home therapy.

An Overview on Principles for Energy Efficient Robot Locomotion.

Despite enhancements in the development of robotic systems, the energy economy of today's robots lags far behind that of biological systems. This is in particular critical for untethered legged robot locomotion. To elucidate the current stage of energy efficiency in legged robotic systems, this paper provides an overview on recent advancements in development of such platforms. The covered different perspectives include actuation, leg structure, control and locomotion principles. We review various robotic actuators exploiting compliance in series and in parallel with the drive-train to permit energy recycling during locomotion. We discuss the importance of limb segmentation under efficiency aspects and with respect to design, dynamics analysis and control of legged robots. This paper also reviews a number of control approaches allowing for energy efficient locomotion of robots by exploiting the natural dynamics of the system, and by utilizing optimal control approaches targeting locomotion expenditure. To this end, a set of locomotion principles elaborating on models for energetics, dynamics, and of the systems is studied.