Lecture 2: Cellular Microenvironment & Extracellular Matrix PDF

Lecture-2: Cellular microenvironment & extra-cellular matrix Outline: - Cellular microenvironment - Matrix components - 3D organization of the matrix components - Physico-chemical properties of the matrix and ageing  Cell...

Lecture-2: Cellular microenvironment & extra-cellular matrix Outline: - Cellular microenvironment - Matrix components - 3D organization of the matrix components - Physico-chemical properties of the matrix and ageing  Cellular Microenvironment DOI:10.1021/acs.chemrev.7b00094  Proteins that sense the cellular microenvironment Various proteins that have been discussed as potential sensors in different cells are displayed. These proteins have been proposed to be activated by membrane stretch or by fluid shear stress. Possible sensors: - PLCβ, phospholipase Cβ; - PLA2, phospholipase A2; - AA, arachidonic acid; - AC, adenylyl cyclase; - PIP2, phosphatidylinositol 4,5-bisphosphate; - IP3, inositol trisphosphate; - DAG, diacylgylcerol; - Src, Src tyrosine kinase; - cAMP, adenosine 3′,5′-cyclic monophosphate; - ATP, adenosine-5′-triphosphate; - Piezo, piezo1 and piezo 2, a new family of mechanosensitive cation channels; - TRP, transient receptor potential; - TREK-1, TWIK-related potassium channel; - Deg, degenerin; - ENaC, epithelial sodium channel. Storch et.al, Americal Journal of Physiology, 2012  Cellular microenvironment and cell-type specific sensing mechanisms: Epithelial and endothelial cells adhere to the basement membrane via only one surface, whereas mesenchymal cells, including fibroblasts, are completely surrounded by the extracellular matrix (ECM). Watt et.al, Nature Reviews Molecular Cell Biology 14, 467–473 (2013) Extra-cellular matrix components: Proteoglycans (PGs) are composed of glycosaminoglycan (GAG) chains covalently linked to a specific protein core (with the exception of hyaluronic acid). PGs have been classified according to their core proteins, localization and GAG composition. The three main families are: small leucine-rich proteoglycans (SLRPs), modular proteoglycans and cell-surface proteoglycans. These molecules are extremely hydrophilic and, accordingly, adopt highly extended conformations that are essential for hydrogel formation and that enable matrices that are formed by these molecules to withstand high compressive forces. Many genetic diseases have been linked to mutations in PG genes. SLRPs have been involved in multiple signaling pathways including binding to and activation of epidermal growth factor receptor (EGFR), insulin-like growth factor 1 receptor (IGFIR) and low-density lipoprotein-receptorrelated protein 1 (LRP1), regulation of inflammatory response reaction, binding to and activation of TGFb. Modular PGs can modulate cell adhesion, migration and proliferation. Basement membrane modular PGs (perlecan, agrin and collagen type XVIII) have a dual function as pro- and anti-angiogenic factors. doi:10.1242/jcs.023820 Cell-surface PGs (syndecans and glypicans) can act as co-receptor facilitating ligand encounters with signaling receptors. Extra-cellular matrix components (continued): The main fibrous ECM proteins are collagens, elastins, fibronectins and laminins More than 28 types of collagen have been identified in vertebrates. The majority of collagen molecules form a triple-stranded helix that subsequently can assemble into supramolecular complexes, such as fibrils and networks, depending on the type of collagen. Fibrous collagens form the backbone of the collagen fibril bundles within the interstitial tissue stroma, whereas network collagens are incorporated into the basal membrane (BM). Collagen associates with elastin, another major ECM fiber. Elastin fibers provide recoil to tissues that undergo repeated stretch. Importantly, elastin stretch is crucially limited by tight association with collagen fibrils Cell-surface binding of the soluble fibronectin (FN) dimer is essential for its assembly into longer fibrils. Moreover, cell contraction through the actomyosin cytoskeleton and the resulting integrin clustering promotes FN– fibril assembly by exposing cryptic binding sites, thus allowing them to bind one another. Like FN, other ECM proteins such as tenascin exert pleiotrophic effects on doi:10.1242/jcs.023820 cellular behavior, including the promotion of fibroblast migration during wound healing Schematic overview of extracellular matrices, their major components, and cell surface receptors. ECMs are classified into two major types, the interstitial and pericellular matrices. Basement membrane, a type of pericellular matrix, is found between epithelial cells and connective tissue. This layer is composed of a collagen IV network that associates with ECM components including nidogen, laminin, perlecan, and minor collagens like collagen XV and XVIII. Epithelial cells are anchored to basement membranes by hemidesmosomes formed via interactions of integrins with laminins. Interstitial matrices are composed of collagen fibrils, elastin, secreted PGs and HA, and matricellular proteins. They interact with each other creating a dynamic and complex three-dimensional network. Cells bind to ECM components by specific cell surface receptors, such as integrins; cell surface PGs; syndecans and glypicans; the HA receptor CD44; and DDRs. They transduce signals into cells that regulate various cellular functions. Growth factors (GF) are sequestered within ECM via binding to ECM components like PGs. They are liberated following ECMdegradation bind to specific growth factor receptors (GF-R) and co-receptors (syndecans and glypicans) and activate various signaling pathways. Several proteolytic enzymes, such as MMPs, ADAMs, ADAMTs, cathepsins, and plasminogen activators, and GAG-degrading enzymes, such as heparanases and hyaluronidases degrade ECMs. Intracellular proteoglycan serglycin is also present in the cytoplasmin to secretory granules in association with bioactive molecules, such as proteases and is secreted in ECM during inflammation and tissue remodeling. ECM degrading enzymes play critical roles in normal tissue remodeling and disease progression. http://dx.doi.org/10.1016/j.addr.2015.11.001  Mechanical interactions between cells and extracellular matrices. Cells interact with ECMs mechanically, including by pulling, often through actomyosin-based contractility coupled to the ECM through integrin-based adhesions, and by pushing, often through actin polymerization and microtubules. The mechanical properties of ECMs mediate these interactions, resulting in cell mechanotransduction and affecting cell behaviours. https://doi.org/10.1038/s41586-020-2612-2  Schematic diagram showing the key mechanical components of the extracellular matrix (ECM) which surrounds neurons in the brain. 1. Neural interstitial matrix 2. Perineuronal net 3. Astrocyte-blood contacts 4. Tripartite synapses 5. Cytoskeleton 6. Synaptic junctions https://doi.org/10.1111/ejn.14766 Sensing extracellular matrix (ECM): from single molecules to tissues. (a) A major research challenge involves understanding how tensile forces switch the structure–function relationship of proteins and how this regulates the reciprocal cross talk between ECM and cells at the organ level. (b) Making advances requires developing atomistic models that can predict how the structure–function relationship of proteins is switched when stretched into intermediate states. (c) Experimental validations require the development of numerous new stretch-sensitive probes that can be applied to read out ECM fiber strain in cell culture and at the organ level. Such probes will greatly advance our knowledge on how to exploit the mechanobiology of ECM for diagnostic, regenerative, and therapeutic applications. (d) The landscape by which the mechanobiology of ECM fibers regulates cell functions is complex. Forces can tune cell and tissue functions through a diverse set of pathways, many of which can cross talk to each other. (e) Although rigidity sensing has been extensively discussed, other feedback loops by which the mechanical switching of ECM protein functions can regulate cell and tissue functions have received far less attention. https://doi.org/10.1146/annurev-physiol-021317-121312  Fluorescence based sensors to detect extra-cellular matrix physical properties Exploiting FRET-labeled FN as a mechanical strain sensor. Overview showing the cryptic cysteines on the FN type III7 and III15 modules and the FRET-labeling scheme. FN isolated from human blood is thereby labeled in vitro and added to the cell medium, which allows cells to harvest and incorporate it into their newly assembled ECM. Note that less than 10% of FN should be FRET-labeled to prevent intermolecular energy transfer. Abbreviations: Cys, cysteine; ECM, extracellular matrix; FN, fibronectin; FRET, fluorescence resonance energy transfer; Lys, lysine; M, molar; RGD, arginine-glycine-aspartic acid. https://doi.org/10.1146/annurev-physiol-021317-121312  Human degradome and schematic representation of major extracellular matrix proteases. - The human degradome is represented by at least 569 proteases distributed intra- and extracellularly, as well as at the cell membrane and subdivided into five families: - metalloproteases - serine proteases - cysteine proteases - aspartic acid proteases - threonine proteases. - In the right panel, a schematic representation of the variable structural protease domains is depicted. - All proteases are basically expressed consisting of a signal sequence, a propeptide, that is cleaved upon activation, and a catalytic domain. - However, several other structural domains distinguish the proteases families between each other, apart from the chemical moiety that participates in the hydrolysis, characterizing their localization and unique interactions. http://dx.doi.org/10.1016/j.addr.2015.11.001 Mechanical behaviours relevant to biological tissues and extracellular matrices a, In linear elastic materials, stress (σ) is linearly related to strain (ε) for small strains (σ = Eε; where E is the elastic modulus), with no loss of mechanical energy and reversible deformations (that is, the loading and unloading curves follow the same path). b, In nonlinear elastic materials, stress is nonlinearly related to strain for even small strains (σ = E(ε)ε). c, Viscoelastic materials exhibit a combination of storage of elastic energy, as a solid, and loss of mechanical energy, as a fluid. This is reflected by hysteresis in the stress–strain relationship during loading and unloading (σ = E(t)ε). The ratio of loss to storage is dependent on time. Viscoelastic materials exhibit stress relaxation in response to a constant deformation, and increased strain, or creep, in response to a constant stress. d, Poroelastic materials exhibit a time-dependent mechanical response due to water flow into or out of a porous network when a deformation induces change in volume (σ = E(ΔV, t)ε; where ΔV is the change in volume). e, Mechanical plasticity refers to the irreversible deformation of a material following application of mechanical loading (ε(t∞) > 0). https://doi.org/10.1038/s41586-020-2612-2  Biological tissues and extracellular matrices are viscoelastic and exhibit stress relaxation in response to a deformation. a, Plot of the loss modulus at approximately 1 Hz (which is a measure of viscosity or dissipation) versus the storage modulus at approximately 1 Hz (which is a measure of elasticity) for skeletal tissues, soft tissues and reconstituted ECMs. The grey dotted line indicates a loss modulus that is 10% of the storage modulus. Shear storage and loss moduli were converted to storage and loss moduli by assuming a Poisson ratio of 0.5, and thus multiplying by a factor of 3. b, Stress relaxation tests on the indicated tissues. Data for a and b result from various modalities of measurement (shear, compression, tension), various measurement tools (mechanical testers, nanoindentation, atomic force microscopy, shear rheometry), and tissue of different animal origins (human, rat, mouse, bovine, sheep, porcine, canine). https://doi.org/10.1038/s41586-020-2612-2 Alterations in the extra-cellular matrix structure and function with ageing epithelium: - Degradation of the collagen matrix - Degenerated elastin network - Increased MMP activity - Altered mechanical responses - …. doi:10.1242/jcs.023820 ECM structures and cellular components in the aortic wall Young aortic wall (left): In intima, endothelial cells (ECs) maintain homeostasis of the wall by forming seamless barrier structure over the basement membrane (BM) and producing vasoprotective factors such as nitric oxide (NO). In media, key ECM molecules (e.g., elastin, collagen) and vascular smooth muscle cells (VSMCs) create the contractile units to maintain vascular tone and compliance. In adventitia, collagen fibers support the aortic wall to prevent overexpansion, and various adventitial cellular and non-cellular ECM components maintain homeostasis of the aortic wall. Aged aortic wall (right): Aging induces senescence of the ECs, which leads to chronic low-grade inflammation and subsequent aberrant ECM remodeling (fragmentation of elastin, excess deposition of collagen and their crosslinking) in the intima and media. Adventitial fibroblasts directly or indirectly stiffen the aortic wall by depositing excessive collagens. https://doi.org/10.3389/fcell.2022.822561 Schematic representation of dermis evolution during human development and ageing. https://doi.org/10.1016/j.mad.2018.03.006 Stromal deregulation in the aged microenvironment drives tumorigenesis and progression. https://doi.org/10.1038/s4 1568-019-0222-9  Age- induced contextual changes in extracellular matrix structure and function in the tumour microenvironment. https://doi.org/10.1038/s4 1568-019-0222-9  Lecture-2: Cellular microenvironment & extra-cellular matrix Summary: - Cellular microenvironment - Matrix components - 3D organization of the matrix components - Physico-chemical properties of the matrix and ageing - Actin - Microtubules - Intermediate filaments Source: Wikipedia Lecture-3: Cell morphometric changes & cytoskeletal remodeling Typical papers for presentation (in 2022): Paper-1: Paper-2: Paper-2: Final exam: Lecture-1: Hallmarks of cellular ageing Lecture-2: Cellular microenvironment & extra-cellular matrix Lecture-3: Cell morphometric changes & cytoskeletal remodeling Lecture-4: Proteostasis Lecture-5: Mitochondrial dysfunction Multiple choice questions: Lecture-6: Endo-membrane signaling Lecture-7: Nuclear signaling & epigenetic alternations 2 questions/lecture = 24 questions Lecture-8: Chromatin remodeling & gene expression Lecture-9: Genomic integrity Answer any 20 questions Lecture-10: Ageing cell secretome and cellular homeostasis Lecture-11: Diseases associated with cellular ageing Lecture-12: Cellular rejuvenation strategies Lecture-13: Therapeutic interventions to cellular ageing Lecture-14: Final exam Typical questions (5 marks per question): Q1: Extra-cellular matrix (ECM) and aging a. Young tissues are stiffer than middle aged tissues (Y/N) b. Tissue matrix composition is altered with aging (Y/N) c. Tissue matrix organization is unchanged with aging (Y/N) d. Matrix is elastic and not viscoelastic (Y/N) e. Stiffness of the matrix is similar in all tissues (Y/N) Q2: ECM sensing and aging a. Only the matrix signals are sensed by cells (Y/N) b. Cells always sense both matrix and biochemical signals (Y/N) c. Matrix composition regulate cellular sensing (Y/N) d. Matrix stiffness regulate cellular sensing (Y/N) e. Cells release signals to modulate matrix microenvironment (Y/N)