Lecture 3: Cellular Microenvironment & Extracellular Matrix PDF

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 Lecture-3: Cell morphometric changes & cytoskeletal remodeling Outline: - Cytoskele...

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 Lecture-3: Cell morphometric changes & cytoskeletal remodeling Outline: - Cytoskeletal components and their cross-talk regulate cell morphology - Extra-cellular matrix (ECM) dictates the 3D organization of cytoskeletal components - Cytoskeletal control of nuclear morphology - Cell morphometric and cytoskeletal alterations in ageing Cytoskeletal components and cell morphology - Actin - Microtubules - Intermediate filaments Herrmann et.al, Nature Reviews Molecular Cell Biology 8, 562-573 (July 2007)  Cytoskeletal components Actin Microtubules Intermediate filaments The cytoskeleton is a cellular entity, encompassing a multitude of filamentous proteins, forming structures that impart mechanical strength, allow intracellular transport and spatial organization, connect the cell to its environment, and generate forces that permit movement. https://doi.org/10.1007/978-3-030-25650-0_12 Actin networks organize in distinct architectures and modules in cells (a) Actin organization in a U2OS cell, visualized by fluorescent actin. The actin cytoskeleton organizes into diverse superstructures in cells, including branched networks in the lamellipodium at the cell front, contractile transverse arcs in the lamella behind the lamellipodium, cross-linked and contractile meshworks in the cortex, and stress fibers stretching toward the cell rear. Scale bar represents 10 μm. (b) Actin is a living polymer that utilizes energy from ATP-hydrolysis to assemble monomers at the barbed end and to disassemble them from the pointed end. By associating with specific binding partners, actin can assemble the diverse architectures seen in panel a. (c) Flow of information toward actin. Extracellular cues are integrated by membrane receptors to activate signaling pathways, including those that regulate the assembly of actin structures. As an example, here we show the Rho pathway that synchronizes the assembly of the actomyosin system through formin-mediated actin polymerization and myosin II–driven contractility. https://doi.org/10.1146/annurev-conmatphys-031218-013231  Cross-talk between actin and microtubule cytoskeleton Mechanisms of actin–microtubule crosstalk. a | Guidance of microtubule growth. Actin–microtubule crosslinking proteins that associate with growing microtubule ends via microtubule plus-end trackers (+TIPs) provide dynamic links between microtubules and actin bundles, which can redirect microtubule growth along actin bundles. b | Anchoring and stabilization of microtubule ends. Protein complexes associated with cortical actin networks can capture both the plus and minus ends of microtubules, leading to stable connections between the two cytoskeletal systems. In the example shown, a plus end is stabilized by a complex involving a motor protein, but the composition of the complex may vary depending on the context. c | Actin as a physical barrier for microtubule growth. The actin cortex may act as a physical barrier that prevents growing microtubules from reaching the plasma membrane by blocking growth and inducing catastrophes. d | Nucleation of actin filaments at microtubule ends. Actin nucleation factors such as formins may associate with growing microtubule ends, which leads to microtubule-mediated local stimulation of actin polymerization. f | Mechanical cooperation in membrane protrusions. Stiff microtubules may provide mechanical support against membrane retraction in events of membrane protrusion driven by actin polymerization. This mechanical support leads to cooperative behaviour of the actin and microtubule cytoskeletons in cell motility. GAP, GTPase-activating protein; GEF, guanine nucleotide exchange factor ; MAP, microtubule-associated protein. Dogterom, M., & Koenderink, G. H. (2019). Nature Reviews Molecular Cell Biology, 20(1), 38-54.  Shared regulators of actin and microtubule dynamics Dogterom, M., & Koenderink, G. H. (2019). Nature Reviews Molecular Cell Biology, 20(1), 38-54. e | Members of the RHO family of small GTPases regulate both actin and microtubule dynamics via their interaction with both actin and microtubule-associated proteins. In addition, microtubules may contribute to the local regulation of actin dynamics via their influence on RHO GTPase activity.  Cross-talk between actin and microtubule cytoskeleton at focal adhesion (A) Focal adhesions link the extracellular matrix via transmembrane receptors (integrins) via talin (yellow) to actin filaments (orange). Vinculin (blue) binds talin and actin and reinforces tension within focal adhesions. Recycling and new delivery of integrins and other adhesion receptors depend on directional transport on microtubules using KIF1C and dynein. Guidance of microtubule assembly along actin fibres mediated by EB1/EB3 (red) that recognise growing microtubule ends and MACF (blue) that link EBs to actin. EBs also mediate the formation of signalling complexes at microtubule plus ends and deliver relaxation factors to focal adhesions. (B) Microtubules are captured at cortical sites near focal adhesions through a complex involving CLASPs (magenta), LL5β (purple), ELKS and Liprin (blue). These are linked to focal adhesions via Kank proteins (brown). CLASPs stimulate microtubule rescues to keep microtubule ends close to the cortical capture site. APC and mDia (blue) cooperate to nucleate actin filaments and also stabilise microtubules. Microtubules regulate Rho GTPases signalling locally, for example by sequestering GEF-H1, which is activated upon its release. GEF-H1 in turn activates RhoA, which stimulates contractility through myosin II and actin assembly through mDia. Abbreviations: APC, adenomatous polyposis coli; GEF-H1, guanine nucleotide exchange factor-H1. Garcin, C., & Straube, A. (2019). Essays in biochemistry, 63(5), 509-520.  Cell-matrix and cell-cell sensing: common mechanisms Mechanical forces generated by acto-myosin interactions within the cytoskeleton are resisted by integrin adhesions to the ECM, cadherin adhesions to neighboring cells and internal cytoskeletal struts (e.g. microtubules and cross-linked actin bundles as in filopodia), thereby establishing a tensional prestress that stabilizes cell and tissue structure through a tensegrity mechanism. Mammoto et.al, Development 137, 1407-1420 (2010)  Cell adhesion to the matrix shapes the cell nucleus Nascent adhesions (NAs) emerge at the leading edge of cell protrusions by nucleating multiple ligand-bond integrins that have been activated by talin and kindlin. Adhesome proteins such as vinculin are subsequently recruited to adhesion sites via talin in a tension- dependent manner or via paxillin in a tension-independent manner. NAs are dynamically coupled to the polymerizing branched actin network through proteins of the molecular clutch such as talin and vinculin, which convert the retrograde movement of polymerizing branched actin network into a protrusive force at the leading edge membrane and Zhiqi Sun et al. J Cell Biol 2016;215:445-456 rearward traction force on the ECM.  Extra-cellular matrix control of nuclear morphology and position Mechanical stimulation from the extracellular matrix is received by mechanosensors such as integrins within the plasma membrane. Force transmission alters the organization of cytoskeletal networks of actin, microtubules and intermediate filaments. These factors either directly or indirectly (through proteins such as plectin) connect to nesprins, components of the LINC complex that spans the nuclear envelope. Nesprins (KASH domain proteins) associate with SUN domain proteins within the lumen between the inner and outer nuclear membranes. SUN domain proteins interact with lamins, a major component of the nuclear lamina that lines the inner side of the nuclear envelope. The lamina also contains LEM domain proteins, including emerin, LAP2beta, and LBR. LEM domain proteins interact with the chromatin binding protein BAF to organize the genome. Thus, mechanical stimulation is transmitted through a cell by a complex network of proteins that terminate in the nucleus and alter gene expression, allowing the cell to respond to its environment. https://doi.org/10.1016/j.gde.2016.03.007  Summary of the various links between inner nuclear membrane to cytoskeleton interactions  Experiments to demonstrate prestressed nuclear architecture in living cells CytoD: actin depolymerizing agent Noc: Microtubule depolymerizing agent Blebb: Myosin motor inhibition IsoNuc: Isolated nucleus Cont: Control cell nucleus force balance Mazumder et.al. J R Soc Interface. (2010) Suppl 3:S321-30  Cytoskeleton organization is dependent on cell microenvironment interaction - Fibroblasts http://www.cellmigration.org/topics/adhesion.shtml  Fragmentation of collagen fibrils within dermis of aged/photoaged skin causes collapse of fibroblasts. Human dermal tissue C. D. A. Extra-cellular matrix Young dermal fibroblast B. Old dermal fibroblast A) Transmission electron micrograph of fibroblast (artificially colored pink for clarity) within dermis of young adult, sun-protected human skin. B) Transmission electron micrograph of fibroblast (artificially colored for clarity) within dermis of photodamaged human skin. (C) Scanning electron doi: 10.1001/archderm.144.5.666 micrograph of collagen fibrils in young & old adult human skin. D) Model depicting relationship between mechanical tension, and collagen production and fragmentation in human skin. The proposed model for the potential mechanism of RhoA/Sun2- mediated increase of cytoskeletal stiffness in progeria cells. The ECM, cytoskeleton and nucleoskeleton are physically connected. Results suggest that both increased ECM stiffness and nuclear stiffness of progeria cells contribute to increased RhoA activation and cytoskeleton stiffness. Also, elevated ROS production, inflammatory signaling (NF-κB), and DNA damages in progeria cells can promote RhoA activation, resulting in further increased cytoskeleton stiffness. Increased RhoA and Sun2 expression, nuclear blebbing, and cytoplasmic DNA fragments in micronuclei, can all mediate accelerated cellular senescence. https://doi.org/10.1111/acel.13152  Cellular senescence is associated with reorganization of the microtubule cytoskeleton. Summary of the cytoskeletal changes in senescent epithelial cells. Senescence of renal proximal tubule cells is associated with the depletion of Histone Deacetylase (HDAC6), Rock1 and γ-tubulin. This results in functional changes to the cytoskeleton. Microtubules are stabilized and cell migration is impaired. https://doi.org/10.1007/s00018-018-2999-1  Imbalanced nucleo-cytoskeletal connections create common polarity defects in progeria and physiological aging Representative images of microtubules (MT) and nuclei (DAPI) in WT (Left) and HGPS (Right) fibroblasts treated with 10 μM nocodazole for 2 h. Inverted contrast is shown. Nocodazole-resistant microtubules surrounding nuclei (arrows) were observed more frequently in HGPS fibroblasts Model for the imbalanced engagement of the nucleus compared with WT fibroblasts. with the cytoskeleton in fibroblasts from Hutchinson– Gilford progeria syndrome (HGPS) and aged individuals. https://doi.org/10.1073/pnas.1809683116  Biological age-dependent changes of cell morphology Traction force A: Schematic defining of single cell morphology. microscopy B&C: Representative immunofluorescence confocal microscopy of young (age < 5) and old (age > 80) human dermal fibroblasts (HDFs) displaying F- actin (green) and nucleus (DAPI, blue). D, E & F: Old cells are larger (D,E) and less elongated (F) than young cells. Cell aspect ratio is defined as W/L, where L is the longest chord of the cell and W is the caliper width, perpendicular to the length, approaching 1 for a less elongated and relatively symmetric cell shape and 0 for an elongated cell shape. doi:10.3390/mi11090801  Age-dependent alteration of nuclear morphology (A–H) Morphometric comparison of nuclear morphology between young (age < 5) and old (age > 80) HDFs. Schematic defining of descriptors of nuclear morphology (A). Note that shape factor is defined as 4π(area)/(perimeter)2, and thus, the nucleus shape factor of elongated and round nuclei approaches 0 and 1, respectively. Representative immunofluorescent images of nuclei of young and old HDFs stained with DAPI (B,C). Nuclei of young cells are smaller (D–G) and more elongated (H) than nuclei of old cells. doi:10.3390/mi11090801  Age-dependent alteration of cell motility Reduced migration doi:10.3390/mi11090801 G: Schematic definition of single cell motility illustrates cell trajectory (orange), persistence vectors (red), and end-to-end distance (blue) of HDFs migrating on a type-I rat-tail collagen coated glass coverslips. H&I: Representative cell trajectories of young (H) and old HDFs (I) displayed distinct cell migration depending on cellular age. J,K &L: Systematic cell tracking based on time-lapse cell monitoring reveal that biological aging significantly reduces cell migrating speed (J) and migratory persistence (K,L). Note that average speed is the total distance traveled by the cell for 8 h of total monitoring time (J), indicating instantaneous motility of the cell, while persistence distance assessed by an average length of persistence vectors (K) and end-to- end distance (L) implies the persistence of cell migration. Heterogeneity in cellular mechanical responses with ageing https://doi.org/10.1038/s41551-017-0093  Heterogeneity in cellular functional responses with ageing https://doi.org/10.1111/nyas.14529 Lecture-3: Summary - Cytoskeletal components and their cross-talk regulate cell morphology - Extra-cellular matrix (ECM) dictates the 3D organization of cytoskeletal components - Cytoskeletal control of nuclear morphology - Cell morphometric and cytoskeletal alterations in ageing Lecture-4: Proteostasis Lopez- Otin, C., et al., Cell 153, 1194–1217 (2013).