CSB 331 University of Michigan Advanced Cell Biology Poster

Welcome to CSB331H1: Advanced Cell Biology Instructors: Prof. Sergey Plotnikov [email protected] Prof. Tony Harris [email protected] Teaching Assistants: Rebecca Tam [email protected] Fernando Valencia [email protected] Ernest Iu [email protected] Microscopy by Ernest Iu 1 CSB331H1 Advanced Cell Biology: General course information Please see syllabus on Quercus for full details 2 CSB331H1 Advanced Cell Biology: General course information Classes: Mondays from 2:10pm to 3pm and Wednesdays from 2:10pm to 4pm Sidney Smith Hall, Room 2117 **Classes will be in-person only. **Classes will not be recorded by instructors. **Copies of the slides will be provided on Quercus. 3 CSB331H1 Advanced Cell Biology: General course information Q&A During lectures by raising your hand. During designated class time by raising your hand. Through Quercus “Discussions”. During Office Hours: Fridays 4-5pm (or by appointment) at instructor office or by Zoom (see syllabus). 4 CSB331H1 Advanced Cell Biology: General course information Grading: First half (Harris) 15% – Poster (submitted through Quercus by January 27) 5% – Pre-Class Quizzes on Research Papers (completed on Quercus while reading the papers before the class) 30% – Mid-Term Test (during class time on February 15; 2h) Second half (Plotnikov) 10% – Three In-Class Quizzes on Research Papers (completed on Quercus; see the course schedule for details) 10% – Written Assignment (one-page assignment on a research paper; submitted through Quercus on date to be announced) 30% – Final Assessment (the format and date/time to be announced; 2 h) 5 CSB331H1 Advanced Cell Biology: General course information Reading: Review articles and free online textbooks provide supporting background information (see syllabus and below) Research articles are critical to read in detail Day 1 reading: Rohn JL, Baum B. Actin and cellular architecture at a glance. J Cell Sci. 2010 Jan 15;123(Pt 2):155-8. doi: 10.1242/jcs.049759. PMID: 20048336. Radisky DC. Epithelial-mesenchymal transition. J Cell Sci. 2005 Oct 1;118(Pt 19):4325-6. doi: 10.1242/jcs.02552. PMID: 16179603. Day 2 reading: St Johnston D. Establishing and transducing cell polarity: common themes and variations. Curr Opin Cell Biol. 2018 Apr;51:33-41. doi: 10.1016/j.ceb.2017.10.007. PMID: 29153703. 6 Main questions How is cell and tissue structure controlled for animal development and physiology? What are the underlying molecular and physical mechanisms? 7 Main questions How is cell and tissue structure controlled for animal development and physiology? What are the underlying molecular and physical mechanisms? Main goals Learn concepts and techniques of cell biology Learn how experiments are designed and interpreted Gain experience at reading primary research articles 8 First half Day 1 Actin network assembly to shape cells Day 2 Polarization of the cell cortex for spatial control of activities Day 3 Cell-cell adhesion to form epithelia Day 4 Epithelial morphogenesis for animal development Background Concepts and Case Studies * Poster due on January 27 (based on Days 1-4) * Quiz completed while reading research article before Day 6 Days 5-6 Forming the first embryonic tissue: background and research article * Quiz completed while reading research article before Day 8 Days 7-8 Tissue internalization: background and research article * Quiz completed while reading research article before Day 10 Days 9-10 Tissue growth control: background and research article Day 11 Review Day 12 *Test 9 First half Day 1 Actin network assembly to shape cells Day 2 Polarization of the cell cortex for spatial control of activities Day 3 Cell-cell adhesion to form epithelia Day 4 Epithelial morphogenesis for animal development Background Concepts and Case Studies * Poster due on January 27 (based on Days 1-4) * Quiz completed while reading research article before Day 6 Days 5-6 Forming the first embryonic tissue: background and research article 1 * Quiz completed while reading research article before Day 8 Days 7-8 Tissue internalization: background and research article 2 * Quiz completed while reading research article before Day 10 Days 9-10 Tissue growth control: background and research article 3 Day 11 Review Day 12 *Test Background and Research Articles 10 First half Day 1 Actin network assembly to shape cells Day 2 Polarization of the cell cortex for spatial control of activities Day 3 Cell-cell adhesion to form epithelia Day 4 Epithelial morphogenesis for animal development * Poster due on January 27 (based on Days 1-4) * Quiz completed while reading research article before Day 6 Days 5-6 Forming the first embryonic tissue: background and research article 1 * Quiz completed while reading research article before Day 8 Days 7-8 Tissue internalization: background and research article 2 * Quiz completed while reading research article before Day 10 Days 9-10 Tissue growth control: background and research article 3 Day 11 Review Day 12 *Test 11 First half Mondays Wednesdays Lecturing Lecturing Group discussion Q Group discussion Q Lecturing Lecturing Group discussion Q Group discussion Q Lecturing Lecturing Q&A 12 How do cells and tissues change their structure during animal development and physiology? Zebrafish cells dividing, adhering, and spreading to form an early embryo tissue Zebrafish leukocytes migrating to a wound site (top) CIL:35272, Danio rerio, embryonic cell. CIL. Dataset (cellimagelibrary.org) CIL:35187, Danio rerio, leukocyte. CIL. Dataset (cellimagelibrary.org) 13 Conventional human-made structures are made from passive materials by external machines Cells and tissues are made from active/smart materials by self-organization 14 “Smart materials are defined as materials that sense and react to environmental conditions or stimuli (e.g., mechanical, chemical, electrical, or magnetic signals)” Smart Material – an overview | ScienceDirect Topics Synthetic smart materials are being developed for a range of applications: – drug delivery and medical devices – responsive textiles and self-cleaning surfaces – sensors, data processing, and robotics – plus many more Elasticity and stability of shape-shifting structures – ScienceDirect The actin cytoskeleton is a natural smart material 15 The basics of actin polymerization (a reminder from BIO230) Actin monomers (G-actin) are asymmetric and bind ATP (red) or ADP (orange) Actin monomers assemble head-to-tail forming filaments with a plus end and a minus end *Asymmetry allows directional growth and directional displacement by motors* ATP-bound monomers preferentially assemble onto the plus end After a monomer joins the filament, its bound ATP hydrolyzes to ADP ADP-bound monomers are lost from the minus end After monomers are lost from the filament, they are bound by proteins that drive the exchange of ADP for ATP Actin and cellular architecture at a glance | Journal of Cell Science | The Company of Biologists 16 The basics of actin polymerization (a reminder from BIO230) Actin monomers (G-actin) are asymmetric and bind ATP (red) or ADP (orange) Actin monomers assemble head-to-tail forming filaments with a plus end and a minus end *Asymmetry allows directional growth and directional displacement by motors* ATP-bound monomers preferentially assemble onto the plus end After a monomer joins the filament, its bound ATP hydrolyzes to ADP ADP-bound monomers are lost from the minus end After monomers are lost from the filament, they are bound by proteins that drive the exchange of ADP for ATP *Turnover allows redeployment* Actin and cellular architecture at a glance | Journal of Cell Science | The Company of Biologists 17 Basic control of the material: factors affecting polymerization and polymer organization Proteins bind to actin filaments to… control their length, promote their turnover, crosslink them into larger meshes or bundles. Actin and cellular architecture at a glance | Journal of Cell Science | The Company of Biologists 18 Basic control of the material: factors affecting polymerization and polymer organization New actin production is promoted by Rho family small G proteins that activate nucleation promoting factors (NPFs) to activate… …the Arp2/3 complex which binds the side of a pre-existing actin filament and nucleates a new filament from the site (creating branching filaments) …or formins which nucleate filaments and promote filament elongation at the plus end (creating linear filaments) Mechanics of the cellular actin cortex: From signalling to shape change – ScienceDirect Actin and cellular architecture at a glance | Journal of Cell Science | The Company of Biologists 19 Basic control of the material: factors affecting polymerization and polymer organization New actin production is promoted by Rho family small G proteins that activate nucleation promoting factors (NPFs) to activate… …the Arp2/3 complex which binds the side of a pre-existing actin filament and nucleates a new filament from the site (creating branching filaments) …or formins which nucleate filaments and promote filament elongation at the plus end (creating linear filaments) Mechanics of the cellular actin cortex: From signalling to shape change – ScienceDirect Actin and cellular architecture at a glance | Journal of Cell Science | The Company of Biologists 20 Basic effects of the material: generation of pushing forces Branched actin networks can be resolved by electron microscopy Mechanics of the cellular actin cortex: From signalling to shape change – ScienceDirect Arp2/3 complex and actin depolymerizing factor/cofilin in dendritic organization and treadmilling of actin filament array in lamellipodia – PubMed (nih.gov)21 Basic effects of the material: generation of pushing forces Branched actin networks can be resolved by electron microscopy Mechanics of the cellular actin cortex: From signalling to shape change – ScienceDirect and polymerize against the plasma membrane to drive the cell front forward DIC imaging of a zebrafish epidermal cell CIL:37332, epidermal cell. CIL. Dataset (cellimagelibrary.org) Arp2/3 complex and actin depolymerizing factor/cofilin in dendritic organization and treadmilling of actin filament array in lamellipodia – PubMed (nih.gov)22 Basic effects of the material: generation of pulling forces Rho small G proteins also activate myosin motors which pull on actin filaments to generate contractile force Mechanics of the cellular actin cortex: From signalling to shape change – ScienceDirect 23 Basic effects of the material: generation of pulling forces Expansion and concatenation of non-muscle myosin IIA filaments drive cellular contractile system formation during interphase and mitosis – PubMed (nih.gov) Myosin motors can be resolved by super-resolution microscopy (structured illumination microscopy [SIM]) 24 Basic effects of the material: generation of pulling forces Expansion and concatenation of non-muscle myosin IIA filaments drive cellular contractile system formation during interphase and mitosis – PubMed (nih.gov) Myosin motors can be resolved by super-resolution microscopy (structured illumination microscopy [SIM]) and actomyosin networks pull the plasma membrane inward for cell division DIC imaging of an early C elegans embryo CIL:25632, Caenorhabditis elegans, early embryonic cell. CIL. Dataset 25 (cellimagelibrary.org) Actin networks underlie the plasma membrane to form the cell cortex In animal cells, actin networks mainly associate with the plasma membrane Linker proteins (purple) bind to actin filaments and to plasma membrane components (lipids or proteins) In an “inactive”, isolated cell, actin networks form an even, thin material beneath the plasma membrane  the cell cortex Contractility of the actin network and hydrostatic pressure from the cytoplasm create a surface tension over the cell Mechanics of the cellular actin cortex: From signalling to shape change – ScienceDirect Specialized cell surface structure must overcome this surface tension to form 26 Actin networks underlie the plasma membrane to form the cell cortex In animal cells, actin networks mainly associate with the plasma membrane Linker proteins (purple) bind to actin filaments and to plasma membrane components (lipids or proteins) In an “inactive”, isolated cell, actin networks form an even, thin material beneath the plasma membrane  the cell cortex Contractility of the actin network and hydrostatic pressure from the cytoplasm create a surface tension over the cell Mechanics of the cellular actin cortex: From signalling to shape change – ScienceDirect Specialized cell surface structures must overcome this surface tension to form 27 Specialized cytoskeletal structures Actin and cellular architecture at a glance | Journal of Cell Science | The Company of Biologists 28 Specialized cytoskeletal structures Actin and cellular architecture at a glance | Journal of Cell Science | The Company of Biologists Outside signal  Small G protein signal + Plasma membrane attachments  Local actin network assembly + activity “Smart materials are defined as materials that sense and react to environmental conditions or stimuli” 29 Group discussion: How could changes to gene expression affect actin networks and cell structure? 30 How actin networks relate to the functions of cells The contraction of actomyosin networks underlies muscle function and animal movement Physiology of the Muscular System | Basicmedical Key 31 How actin networks relate to the functions of cells The contraction of actomyosin networks underlies muscle function and animal movement The protrusion of actin networks drives neural pathfinding and development of the nervous system Physiology of the Muscular System | Basicmedical Key View Image (nrronline.org) 32 Actin networks are central to the development and maintenance of epithelial tissue organization Epithelia are the most common tissues in animals They form our skin and internal organs They act as regulated barriers ~90% of cancers arise from epithelia large intestine | Definition, Location, Anatomy, Length, Function, & Facts | Britannica 33 Epithelial-mesenchymal transition | Journal of Cell Science | The Company of Biologists Epithelial-to-Mesenchymal Transition Epithelia are based on extensive cell-cell interactions mediated by actin-associated adherens junctions (Day 3) But they can undergo EMT 34 Epithelial-mesenchymal transition | Journal of Cell Science | The Company of Biologists Epithelial-to-Mesenchymal Transition 35 Epithelial-mesenchymal transition | Journal of Cell Science | The Company of Biologists Epithelial-to-Mesenchymal Transition 36 Epithelial-mesenchymal transition | Journal of Cell Science | The Company of Biologists Epithelial-to-Mesenchymal Transition 37 Epithelial-mesenchymal transition | Journal of Cell Science | The Company of Biologists Epithelial-to-Mesenchymal Transition 38 How does the cell organize its surface to deploy molecular activities at specific locations? Actin and cellular architecture at a glance | Journal of Cell Science | The Company of Biologists 1 Definition of “cell polarity” An axis of the cell with distinct molecular activities at each end Can refer to trafficking routes within the cell, 2 Definition of “cell polarity” An axis of the cell with distinct molecular activities at each end Can refer to trafficking routes within the cell, OUT IN Nucleus COPII-coated Vesicle – an overview | ScienceDirect Topics 3 Definition of “cell polarity” An axis of the cell with distinct molecular activities at each end Can refer to trafficking routes within the cell, but typically refers to the cell cortex OUT DOMAIN 1 IN Nucleus DOMAIN 2 4 The least polarized cell? Red blood cell -No nucleus -Uniform cell cortex -No cell division -No cell migration Red Blood Cell – The Definitive Guide | Biology Dictionary (Gradient of oxygen levels between inside and outside could be considered a polarity) 5 Single cell polarity for cell migration Active cell migration involves distinct cytoskeletal activities at the front and rear of the cell Activities at the front: -induction of Arp2/3 network growth -plasma membrane protrusion by actin polymerization -formation of new adhesions to the substrate Activities at the rear: -induction of actomyosin network assembly -plasma membrane contraction by actomyosin activity -detachment of adhesions from the substrate Cytoskeletal Crosstalk in Cell Migration: Trends in Cell Biology 6 Single cell polarity for cell division Cell division involves distinct cytoskeletal activities at the equator and at the poles Activities at the equator: -induction of actomyosin network assembly (blue) -plasma membrane contraction by actomyosin activity Activities at the poles: -thinning of cortical actin network (orange) -plasma membrane protrusions from cytosolic pressure (prevented elsewhere by the thicker cell cortex) Inhibition of polar actin assembly by astral microtubules is required for cytokinesis | Nature Communications 7 Single cell polarity for neural signaling Dendrites receive signals Axons send signals Components of neuron – Neuron – Wikipedia 8 Epithelial apical-basal cell polarity Distinct apical and basal ends of an epithelial cell allow epithelia to act as regulated barriers between body compartments -the apical domain faces the body surface or organ lumen -the basal domain faces the extracellular matrix formed between organs Roles of Cadherins and Catenins in Cell−Cell Adhesion and Epithelial Cell Polarity – ScienceDirect 9 Epithelial apical-basal cell polarity Distinct apical and basal ends of an epithelial cell allow epithelia to act as regulated barriers between body compartments -the apical domain faces the body surface or organ lumen -the basal domain faces the extracellular matrix formed between organs Roles of Cadherins and Catenins in Cell−Cell Adhesion and Epithelial Cell Polarity – ScienceDirect Distinct cytoskeletal networks and intracellular trafficking routes are directed towards the apical and basal domains Adaptation of core mechanisms to generate cell polarity | Nature 10 Epithelial planar cell polarity Epithelial cells can also be polarized across the epithelial sheet Organizes animal development Organizes animal physiology Cells | Free Full-Text | Role of the Polycystins in Cell Migration, Polarity, and Tissue Morphogenesis | HTML (mdpi.com) 11 Epithelial planar cell polarity Planar cell polarity signaling: coordination of cellular orientation across tissues Singh – 2012 – WIREs Developmental Biology – Wiley Online Library Epithelial cells can also be polarized across the epithelial sheet Organizes animal development Organizes animal physiology Cells | Free Full-Text | Role of the Polycystins in Cell Migration, Polarity, and Tissue Morphogenesis | HTML (mdpi.com) 12 Group discussion: In what ways would apical-basal polarity and planar polarity be important to the function of a sensory hair cell of the inner ear? Sensory Hair Cells: An Introduction to Structure and Physiology | Integrative and Comparative Biology | Oxford Academic (oup.com) 13 Fundamental mechanisms of cell polarization (breaking symmetry to make one region of the cell different from another) Positive feedback Repulsion 14 Case study A local positive feedback can recruit factors to a cortical domain Yeast cells polarize to organize budding of a daughter cell Cell Polarity in Yeast | Annual Review of Cell and Developmental Biology (annualreviews.org) 15 A local positive feedback can recruit factors to a cortical domain Yeast cells polarize to organize budding of a daughter cell Cdc42 recruits its activator, the PAK-Bem1-GEF complex, to the bud site Cell Polarity in Yeast | Annual Review of Cell and Developmental Biology (annualreviews.org) 16 A local positive feedback can recruit factors to a cortical domain Yeast cells polarize to organize budding of a daughter cell Cdc42 recruits its activator, the PAK-Bem1-GEF complex, to the bud site Cell Polarity in Yeast | Annual Review of Cell and Developmental Biology (annualreviews.org) Light induced accumulation of a Bem1 protein construct recruits endogenous Bem1 -it also recruits the Cdc42 GEF and Cdc42-GTP -it requires its interaction site with the GEF to do so Bem1 Bem1 TULIPs: tunable, light-controlled interacting protein tags for cell biology | Nature Methods 17 A local positive feedback can recruit factors to a cortical domain Yeast cells polarize to organize budding of a daughter cell Cdc42 recruits its activator, the PAK-Bem1-GEF complex, to the bud site Cell Polarity in Yeast | Annual Review of Cell and Developmental Biology (annualreviews.org) Light induced accumulation of a Bem1 protein construct recruits endogenous Bem1 -it also recruits the Cdc42 GEF and Cdc42-GTP -it requires its interaction site with the GEF to do so Bem1 Bem1 TULIPs: tunable, light-controlled interacting protein tags for cell biology | Nature Methods Cell cycle entry triggers a switch between two modes of Cdc42 activation during yeast polarization | eLife (elifesciences.org) 18 A local positive feedback can recruit factors to a cortical domain Yeast cells polarize to organize budding of a daughter cell Cdc42 recruits its activator, the PAK-Bem1-GEF complex, to the bud site Cell Polarity in Yeast | Annual Review of Cell and Developmental Biology (annualreviews.org) Light induced accumulation of a Bem1 protein construct recruits endogenous Bem1 -it also recruits the Cdc42 GEF and Cdc42-GTP -it requires its interaction site with the GEF to do so Bem1 Bem1 TULIPs: tunable, light-controlled interacting protein tags for cell biology | Nature Methods Cell cycle entry triggers a switch between two modes of Cdc42 activation during yeast polarization | eLife (elifesciences.org) 19 Case study Inhibitory mechanisms can expel factors from a cortical domain Drosophila neuroblasts polarize to asymmetrically divide Polarization of Drosophila Neuroblasts During Asymmetric Division (cshlp.org) 20 Inhibitory mechanisms can expel factors from a cortical domain Drosophila neuroblasts polarize to asymmetrically divide Phosphorylation of Lgl by aPKC expels Lgl from one side of the cell Polarization of Drosophila Neuroblasts During Asymmetric Division (cshlp.org) The Par complex directs asymmetric cell division by phosphorylating the cytoskeletal protein Lgl | Nature 21 Inhibitory mechanisms can expel factors from a cortical domain Drosophila neuroblasts polarize to asymmetrically divide Phosphorylation of Lgl by aPKC expels Lgl from one side of the cell Polarization of Drosophila Neuroblasts During Asymmetric Division (cshlp.org) aPKC phosphorylates Lgl, and three Serine residues of Lgl are required +[γ-32P]-labelled ATP Autoradiograph Coomassie stain The Par complex directs asymmetric cell division by phosphorylating the cytoskeletal protein Lgl | Nature 22 Inhibitory mechanisms can expel factors from a cortical domain Drosophila neuroblasts polarize to asymmetrically divide Phosphorylation of Lgl by aPKC expels Lgl from one side of the cell Polarization of Drosophila Neuroblasts During Asymmetric Division (cshlp.org) aPKC phosphorylates Lgl, and three Serine residues of Lgl are required +[γ-32P]-labelled ATP An Lgl construct lacking the three Serine residues accumulates around the whole cell and causes mis-localization of endogenous proteins Autoradiograph Coomassie stain The Par complex directs asymmetric cell division by phosphorylating the cytoskeletal protein Lgl | Nature 23 Excitable media forms from a combination of local positive feedback and induction of longer range inhibition In the early 1950s, Alan Turing showed mathematically that patterns can arise from such “reaction-diffusion systems” Alan Turing’s Patterns in Nature, and Beyond | WIRED Positional information and reaction-diffusion: two big ideas in developmental biology combine | Development | The Company of Biologists 24 Spatial landmarks can organize patterning mechanisms Instead of the pattern arising spontaneously, patterning mechanisms can be linked to a pre-existing landmark e.g. intrinsic landmarks during cell division Mechanics of the cellular actin cortex: From signalling to shape change – ScienceDirect Spatial information from the spindle and chromosomes organizes RhoA-GTP, actin, and myosin around the cell cortex 25 Spatial landmarks can organize patterning mechanisms e.g. an extrinsic landmark during directed cell migration Neutrophil chemotaxis | SpringerLink Spatial information from a chemotactic gradient of molecules secreted from the target organizes signaling lipids and actin networks around the neutrophil cell cortex 26 Group discussion: Are there examples of these patterning mechanisms (positive feedback or repulsion) beyond cell biology? 27 Molecular mechanisms of epithelial apical-basal polarity 1. Cell junctions as spatial landmarks: Cell-cell junctions mark the lateral plasma membrane domain -Adherens Junctions and Tight Junctions Cell-matrix ju -Integrin adhesions Epithelial cell polarity, stem cells and cancer | Nature Reviews Cancer 28 Molecular mechanisms of epithelial apical-basal polarity 1. Cell junctions as spatial landmarks: Cell-cell junctions mark the lateral plasma membrane domain -Adherens Junctions and Tight Junctions Cell-matrix junctions mark the basal plasma membrane domain -Integrin adhesions Integrins and epithelial cell polarity | Journal of Cell Science | The Company of Biologists 29 Molecular mechanisms of epithelial apical-basal polarity 2. The Crumbs complex and the Par complex interact with each other and cell-cell junctions to define the apical and apicolateral domains (positive feedback by mutual reinforcement): Crumbs complex Par complex Protein Complex Assemblies in Epithelial Cell Polarity and Asymmetric Cell Division – ScienceDirect Epithelial cell polarity, stem cells and cancer | Nature Reviews Cancer 30 Molecular mechanisms of epithelial apical-basal polarity 3. Reciprocal antagonism by phospho-regulation distinguishes the apical/apicolateral domain from the basal/basolateral domain: aPKC phosphorylation of Lgl, Dlg, and PAR-1 displaces Lgl, Dlg, and PAR-1 from the apical/apicolateral domain PAR1 phosphorylation of PAR3 displaces PAR3 from the basal/basolateral domain Epithelial cell polarity, stem cells and cancer | Nature Reviews Cancer 31 Molecular mechanisms of epithelial apical-basal polarity 4. Targeted cytoskeletal assembly, membrane trafficking and barriers to plasma membrane diffusion generate the distinct molecular activities of the apical/apicolateral and basal/basolateral domains: Local actin cytoskeletal assembly produces apical protrusions to increase surface area for molecular exchange, or to detect sounds waves Structure, Regulation, and Functional Diversity of Microvilli on the Apical Domain of Epithelial Cells | Annual Review of Cell and Developmental Biology (annualreviews.org) 32 Molecular mechanisms of epithelial apical-basal polarity 4. Targeted cytoskeletal assembly, membrane trafficking and barriers to plasma membrane diffusion generate the distinct molecular activities of the apical/apicolateral and basal/basolateral domains: Membrane trafficking routes target specific domains and populate them with specific sets of membrane proteins Tight junctions stop lateral diffusion of membrane components between membrane domains, and stop molecular diffusion between cells The epithelial polarity program: machineries involved and their hijacking by cancer | Oncogene (nature.com) 33 Molecular mechanisms of epithelial apical-basal polarity 4. Targeted cytoskeletal assembly, membrane trafficking and barriers to plasma membrane diffusion generate the distinct molecular activities of the apical/apicolateral and basal/basolateral domains: Membrane trafficking routes target specific domains and populate them with specific sets of membrane proteins Tight junctions stop lateral diffusion of membrane components between membrane domains, and stop molecular diffusion between cells Allows control of molecular movement across the epithelium The epithelial polarity program: machineries involved and their hijacking by cancer | Oncogene (nature.com) 34 Roles of Cadherins and Catenins in Cell−Cell Adhesion and Epithelial Cell Polarity – ScienceDirect Molecular mechanisms of epithelial planar cell polarity 1. Developmental signal gradients provide spatial information across the epithelium: A resulting gradient of receptor expression leads to more receptor-receptor interactions at one side of the cell than the other Planar cell polarity: two genetic systems use one mechanism to read gradients | Development | The Company of Biologists 35 Molecular mechanisms of epithelial planar cell polarity 2. Receptor interactions and associated signaling complexes define mutually antagonistic domains on opposite sides of the cell: The cell biology of planar cell polarity | Journal of Cell Biology | Rockefeller University Press (rupress.org) 36 Molecular mechanisms of epithelial planar cell polarity 3. The defined domains direct cytoskeletal and trafficking activities to assemble functional structures at a specific side of the cell: ** the structures are still made with loss of planar cell polarity, but they lose their spatial coordination across the tissue** 37 Planar cell polarity signaling: coordination of cellular orientation across tissues – Singh – 2012 – WIREs Developmental Biology – Wiley Online Library Short break Discussion of poster project Q&A session 38 What bridges scales of organization to form organisms from molecules? hillis2e_ch01 (macmillanhighered.com) What bridges scales of organization to form organisms from molecules? hillis2e_ch01 (macmillanhighered.com) Cell adhesion molecules are key, and can be considered at several levels (with analogies to brick mortar): The structure of cell adhesion molecules ALL YOU WANT TO KNOW ABOUT CEMENT MIX RATIO AND ITS USES lceted LCETED INSTITUTE FOR CIVIL ENGINEERS The linkage of cells by cell adhesion molecules The integration of cells into tissues and organs Basic Bricklaying and Cement-Mixing Guide for Beginners – Dengarden HRCx – Commissioning Consultancy — University of2Toronto Ramsay Wright Teaching Labs Renewal Families of cell adhesion molecules Cell adhesion in cancer: Beyond the migration of single cells – Journal of Biological Chemistry (jbc.org) Common structural features: 1. Protein molecules 2. Extracellular domains 3. Transmembrane domains 4. Cytoplasmic tails Similar to other cell surface receptors 3 Basic mechanisms of cell-cell adhesion Cell-Cell Adhesion – Molecular Biology of the Cell – NCBI Bookshelf (nih.gov) Binding between the same molecules (“Homophilic”) Binding between different molecules (“Heterophilic”) Binding via an extracellular linker protein 4 Basic mechanisms of cell-cell adhesion Cell-Cell Adhesion – Molecular Biology of the Cell – NCBI Bookshelf (nih.gov) Binding between the same molecules (“Homophilic”) Binding between different molecules (“Heterophilic”) Binding via an extracellular linker protein examples: 5 Cell adhesion in cancer: Beyond the migration of single cells – Journal of Biological Chemistry (jbc.org) Examples of cell-cell adhesion in the nervous system Cell adhesion molecules connect axons (fasciculation) to coordinate their growth and organization The role of cell adhesion molecules in visual circuit formation: From neurite outgrowth to maps and synaptic specificity – Missaire – 2015 – Developmental Neurobiology – Wiley Online Library Cell adhesion molecules establish synapses with target cells Cell Adhesion, the Backbone of the Synapse: “Vertebrate” and “Invertebrate” Perspectives (cshlp.org) 6 Examples of cell-cell adhesion in the immune system How leukocytes cross the vascular endothelium | Nature Reviews Immunology Cell adhesion molecules mediate transendothelial migration of white blood cells during an immune response Cell adhesion molecules promote the immune synapse between an Antigen Presenting Cell (APC) and a T cell during T cell activation Orchestrating Lymphocyte Polarity in Cognate Immune Cell–Cell Interactions – ScienceDirect 7 Cell-cell junctions in epithelia Cell-cell junctions in epithelia TEM reveals each junction based on a uniform membrane-membrane distance dependent on the length of the cell adhesion molecule interaction Transmission electron microscopy (TEM) JUNCTIONAL COMPLEXES IN VARIOUS EPITHELIA | Journal of Cell Biology | Rockefeller University Press (rupress.org) 9 Adherens junctions are stabilized by clustering of cadherin adhesion molecules… …and by linkage of cadherins to the actin cytoskeleton via β-catenin and α-catenin PM PM PM 10 The Three-Dimensional Structure of the Cadherin–Catenin Complex | SpringerLink Group discussion: How does the brick and mortar analogy break down? HRCx – Commissioning Consultancy — University of Toronto Ramsay Wright Teaching Labs Renewal CIL:35272, Danio rerio, embryonic cell. CIL. Dataset (cellimagelibrary.org) 11 12 Case study 13 Figure 1. Reconstitution of the Cadherin-Catenin Complex but Not Actin Binding Pelleting assay Centrifuge Large protein complexes (actin networks) pellet Small protein complexes bound to actin networks pellet Free protein complexes remain in supernatant 14 Deconstructing the Cadherin-Catenin-Actin Complex – ScienceDirect Figure 1. Reconstitution of the Cadherin-Catenin Complex but Not Actin Binding Pelleting assay Centrifuge Large protein complexes (actin networks) pellet Small protein complexes bound to actin networks pellet Free protein complexes remain in supernatant β-catenin GST-E-cadherin tail Actin Only actin pellets An E-cadherin-β-catenin complex remains in supernatant 15 Deconstructing the Cadherin-Catenin-Actin Complex – ScienceDirect Figure 1. Reconstitution of the Cadherin-Catenin Complex but Not Actin Binding Pelleting assay Centrifuge Large protein complexes (actin networks) pellet Small protein complexes bound to actin networks pellet Free protein complexes remain in supernatant α-catenin β-catenin GST-E-cadherin tail Actin Actin Only actin pellets α-catenin pellets with actin An E-cadherin-β-catenin complex remains in supernatant The E-cadherin-β-catenin complex remains in supernatant!! 16 Deconstructing the Cadherin-Catenin-Actin Complex – ScienceDirect Figure 3. Reconstitution of the Cadherin-Catenin Complex on Membrane Patches Attach cells to an E-cadherin-coated substrate and “unroof” them to expose the cytoplasmic face of the Cadherin-Catenin Complex 17 Deconstructing the Cadherin-Catenin-Actin Complex – ScienceDirect Figure 3. Reconstitution of the Cadherin-Catenin Complex on Membrane Patches Attach cells to an E-cadherin-coated substrate and “unroof” them to expose the cytoplasmic face of the Cadherin-Catenin Complex Strip peripheral membrane proteins with a chaotropic agent (4 M guanidine hydrochloride) and attempt to re-build 18 Deconstructing the Cadherin-Catenin-Actin Complex – ScienceDirect Figure 3. Reconstitution of the Cadherin-Catenin Complex on Membrane Patches Attach cells to an E-cadherin-coated substrate and “unroof” them to expose the cytoplasmic face of the Cadherin-Catenin Complex Strip peripheral membrane proteins with a chaotropic agent (4 M guanidine hydrochloride) and attempt to re-build α-catenin needs β-catenin to re-associate with the membrane 19 Deconstructing the Cadherin-Catenin-Actin Complex – ScienceDirect Figure 3. Reconstitution of the Cadherin-Catenin Complex on Membrane Patches Attach cells to an E-cadherin-coated substrate and “unroof” them to expose the cytoplasmic face of the Cadherin-Catenin Complex Strip peripheral membrane proteins with a chaotropic agent (4 M guanidine hydrochloride) and attempt to re-build α-catenin needs β-catenin to re-associate with the membrane Actin fails to re-associate when α-catenin, β-catenin, and E-cadherin are there!! 20 Deconstructing the Cadherin-Catenin-Actin Complex – ScienceDirect If this model is true, then something is missing from the reconstitution attempts 21 22 Fig. 2 Cadherin-catenin complexes bound actin filaments in oscillating-stage experiments. A reconstituted E-cadherin tail-β-catenin-α-catenin complex was immobilized on a coverslip via a bead An actin filament was held above the coverslip by optical traps 23 The minimal cadherin-catenin complex binds to actin filaments under force | Science (sciencemag.org) Fig. 2 Cadherin-catenin complexes bound actin filaments in oscillating-stage experiments. The microscope stage was oscillated back-and-forth (gray line in graph) Movement of the stage was expected to drag the Cadherin-Catenin Complex along the actin filament If the applied force resulted in binding between the two, then the actin-bound bead would be pulled from the optical trap Pulling from the trap (converted to blue force measurements in graph) (i) correlated with stage movement and (ii) required α-catenin 24 The minimal cadherin-catenin complex binds to actin filaments under force | Science (sciencemag.org) Group discussion: In what way can the adherens junction be considered a “smart material”? 25 Mechanical signal transduction Signal transduction: The conversion of signal from one form into another to affect cell behavior e.g. an induced protein-protein interaction leading to a downstream phosphorylation event e.g. light exposure leading to a receptor conformational change Mechanical signal transduction: The conversion of a force into a molecular change to affect cell behaviour 26 Forces applied to tissues are distributed among cells and among adherens junctions of the cells [stress (σ)] Tensile Forces and Mechanotransduction at Cell–Cell Junctions – ScienceDirect 27 Forces applied to tissues are distributed among cells and among adherens junctions of the cells Forces from within a cell are also applied to adherens junctions [stress (σ)] Dynamic contacts: rearranging adherens junctions to drive epithelial remodelling | Nature Reviews Molecular Cell Biology Tensile Forces and Mechanotransduction at Cell–Cell Junctions – ScienceDirect 28 Forces can strengthen the cadherin-catenin complex through exposure of cryptic sites Exposed sites can strengthen the α-catenin-actin interaction… Force-dependent allostery of the α-catenin actin-binding domain controls adherens junction dynamics and functions | Nature Communications 29 Forces can strengthen the cadherin-catenin complex through exposure of cryptic sites Exposed sites can strengthen the α-catenin-actin interaction… Property of a “catch bond” (a bond that increases its strength with force) On Modeling Molecular Mechanisms and Mädchenfängers – ScienceDirect Force-dependent allostery of the α-catenin actin-binding domain controls adherens junction dynamics and functions | Nature Communications 30 Forces can strengthen the cadherin-catenin complex through exposure of cryptic sites Exposed sites can strengthen the α-catenin-actin interaction… Force-dependent allostery of the α-catenin actin-binding domain controls adherens junction dynamics and functions | Nature Communications …and can recruit additional actin binding proteins (e.g. vinculin [VCL]) to reinforce α-catenin Dynamic contacts: rearranging adherens junctions to drive 31 epithelial remodelling | Nature Reviews Molecular Cell Biology Forces applied to adherens junctions can also… …(i) bend the plasma membrane, which (ii) recruits curvature sensing BAR domains, which (iii) recruit effectors …and might relay forces that affect mechanosensitive channels Cancers | Free Full-Text | Mechanosensitive Piezo Channels in Cancer: Focus on altered Calcium Signaling in Cancer Cells and in Tumor Progression (mdpi.com) Tensile Forces and Mechanotransduction at Cell–Cell Junctions – ScienceDirect 32 Specific mechanisms downregulate adherens junctions by cadherin endocytosis Adherens junctions: from molecules to morphogenesis | Nature Reviews Molecular Cell Biology 33 Morphogenesis: the generation of form Incredibly detailed embryo maps chart each cell’s developmental fate (nature.com) https://www.nikonsmallworld.com/galleries/2019photomicrography-competition/endothelial-cells-inintestine-of-an-18-5-day-old-mouse-embryo Mouse embryo (day 12.5) stained for motor (red) and sensory (magenta) nerves and nerve endings (cyan) | 2018 Photomicrography Competition | Nikon’s Small World (nikonsmallworld.com) Chameleon embryo | 2018 Photomicrography Competition | Nikon’s Small World (nikonsmallworld.com) Drosophila sp. (fruit fly) larva with the dendrites of a sensory neuron group labeled, live specimen | 2010 Photomicrography Competition | 1 Nikon’s Small World (nikonsmallworld.com) Morphogenesis: the generation of form Basic requirements: Pre-patterns across the tissue (e.g. gene expression patterns) + Pre-patterns within cells of the tissue (cell polarity) Deployment of force generating machinery based on the pre-patterns above 2 Epithelial morphogenesis additionally requires the coordination of force generating machinery with cell-cell adhesion complexes Actin-based force generation and cell adhesion in tissue morphogenesis – ScienceDirect 3 Epithelial morphogenesis additionally requires the coordination of force generating machinery with cell-cell adhesion complexes Force generating machinery must engage cell-cell adhesion complexes at the correct time and place Actin-based force generation and cell adhesion in tissue morphogenesis – ScienceDirect 4 Deployment of actomyosin networks to the apical end of the cell produces apical constriction, wedge-shaped cells, and tissue bending Actin-based force generation and cell adhesion in tissue morphogenesis – ScienceDirect 5 Deployment of actomyosin networks to the apical end of the cell produces apical constriction, wedge-shaped cells, and tissue bending Differences of actomyosin levels between cells produce a distinct shape Actin-based force generation and cell adhesion in tissue morphogenesis – ScienceDirect 6 Deployment of actomyosin networks to specific tissue regions bends the tissue at the sites Folding of the chick hindgut at the caudal intestinal portal (CIP) Pre-pattern of developmental signaling Actin-based force generation and cell adhesion in tissue morphogenesis – ScienceDirect 7 Deployment of actomyosin networks to specific tissue regions bends the tissue at the sites Folding of the chick hindgut at the caudal intestinal portal (CIP) Internalization of the fly mesoderm at the ventral furrow Pre-pattern of developmental signaling Pre-pattern of developmental transcription factor expression Actin-based force generation and cell adhesion in tissue morphogenesis – ScienceDirect 8 Contractile actomyosin networks and expansive Arp2/3 networks have a range of effects On all cell-cell junctions for epithelial bending Actin-based force generation and cell adhesion in tissue morphogenesis – ScienceDirect 9 Contractile actomyosin networks and expansive Arp2/3 networks have a range of effects On all cell-cell junctions for epithelial bending On one cell’s junctions for its removal from the epithelium, or entry into the epithelium Actin-based force generation and cell adhesion in tissue morphogenesis – ScienceDirect 10 Contractile actomyosin networks and expansive Arp2/3 networks have a range of effects On all cell-cell junctions for epithelial bending On one cell’s junctions for its removal from the epithelium, or entry into the epithelium On specific cell-cell junctions for neighbour exchange within the epithelium Actin-based force generation and cell adhesion in tissue morphogenesis – ScienceDirect 11 Group discussion: What needs to occur between the pre-pattern stage and the force-generation stage? 12 Case study Drosophila germband extension/elongation Stages of Drosophila embryogenesis Genetics on the Fly: A Primer on the Drosophila Model System | Genetics 13 Case study: Drosophila germband extension/elongation Dorsal Anterior Posterior Ventral White-light imaging at middle of embryo. Black arrowheads indicate each end of germband. Cell intercalation during Drosophila germband extension and its regulation by pair-rule segmentation genes | Development | The Company of Biologists 14 Germband epithelial cell intercalation (neighbour exchange) is coupled with tissue elongation White-light imaging at surface of embryo. Apical surface of individual cells outlined in black. Cell intercalation during Drosophila germband extension and its regulation by pair-rule segmentation genes | Development | The Company of Biologists 15 Germband epithelial cell intercalation (neighbour exchange) is coupled with tissue elongation (place your hands together and intercalate your fingers for a similar effect) Posterior The cell intercalation is directed along the dorsal-ventral axis and the tissue extends along the anterior-posterior axis Anterior Dorsal Ventral Cell intercalation during Drosophila germband extension and its regulation by pair-rule segmentation genes | Development | The Company of Biologists 16 Germband epithelial cell intercalation (neighbour exchange) is coupled with tissue elongation (place your hands together and intercalate your fingers for a similar effect) Posterior The cell intercalation is directed along the dorsal-ventral axis and the tissue extends along the anterior-posterior axis Anterior Dorsal Ventral What pre-pattern is involved? Cell intercalation during Drosophila germband extension and its regulation by pair-rule segmentation genes | Development | The Company of Biologists 17 Transcription factors of the anterior-posterior patterning system direct the cell intercalation Cell intercalation during Drosophila germband extension and its regulation by pair-rule segmentation genes | Development | The Company of Biologists 18 Transcription factors of the anterior-posterior patterning system direct the cell intercalation Cell intercalation during Drosophila germband extension and its regulation by pair-rule segmentation genes | Development | The Company of Biologists 19 Transcription factors of the anterior-posterior patterning system direct the cell intercalation What force-generating machinery is involved? Cell intercalation during Drosophila germband extension and its regulation by pair-rule segmentation genes | Development | The Company of Biologists 20 Myosin-dependent junctional remodeling is required Cell junctions undergo T1T2T3 transitions 21 Myosin-dependent junction remodelling controls planar cell intercalation and axis elongation | Nature Myosin-dependent junctional remodeling is required Cell junctions undergo T1T2T3 transitions Myosin specifically localizes to the shrinking junction of the T1T2 transition (and is required for the transition [not shown]) T1 T2 T2 T3 T1 T2 T3 T3 22 Myosin-dependent junction remodelling controls planar cell intercalation and axis elongation | Nature Myosin-dependent junctional remodeling is required Cell junctions undergo T1T2T3 transitions Myosin specifically localizes to the shrinking junction of the T1T2 transition (and is required for the transition [not shown]) T1 T2 T2 T3 T1 T2 T3 T3 This planar polarized myosin distribution requires transcription factors of the anterior-posterior patterning system 23 Myosin-dependent junction remodelling controls planar cell intercalation and axis elongation | Nature Myosin-dependent junctional remodeling is required Cell junctions undergo T1T2T3 transitions Myosin specifically localizes to the shrinking junction of the T1T2 transition (and is required for the transition [not shown]) T1 How is myosin recruited to the junction? T2 T2 T3 T1 T2 T3 T3 This planar polarized myosin distribution requires transcription factors of the anterior-posterior patterning system 24 Myosin-dependent junction remodelling controls planar cell intercalation and axis elongation | Nature A canonical signaling pathway is required for the myosin recruitment Mechanics of the cellular actin cortex: From signalling to shape change – ScienceDirect A canonical signaling pathway is required for the myosin recruitment Mechanics of the cellular actin cortex: From signalling to shape change – ScienceDirect Rho kinase (Rok) Interpretation of structure-function analysis Rho GTPase and Shroom direct planar polarized actomyosin contractility during convergent extension | Journal of Cell Biology | Rockefeller University Press (rupress.org) 26 A canonical signaling pathway is required for the myosin recruitment Mechanics of the cellular actin cortex: From signalling to shape change – ScienceDirect Rho kinase (Rok) Interpretation of structure-function analysis The Pleckstrin Homology (PH) domain is required for cortical localization of Rok The Rho Binding (RB) domain is required for planar polarization of Rok Rho GTPase and Shroom direct planar polarized actomyosin contractility during convergent extension | Journal of Cell Biology | Rockefeller University Press (rupress.org) 27 A canonical signaling pathway is required for the myosin recruitment Mechanics of the cellular actin cortex: From signalling to shape change – ScienceDirect Rho kinase (Rok) Interpretation of structure-function analysis The PH domain is sufficient for cortical localization An RB-PH two-domain protein is insufficient for planar polarization An SB (Shroom Binding)-RB-PH three-domain protein is sufficient for planar polarization Rho GTPase and Shroom direct planar polarized actomyosin contractility during convergent extension | Journal of Cell Biology | Rockefeller University Press (rupress.org) 28 A canonical signaling pathway is required for the myosin recruitment Mechanics of the cellular actin cortex: From signalling to shape change – ScienceDirect Rho kinase Loss-of-function studies of the binding partners Rho kinase Abnormal Rho kinase planar polarity in a Shroom mutant Loss of Rho kinase planar polarity with expression of a Rho dominant-negative construct Rho GTPase and Shroom direct planar polarized actomyosin contractility during convergent extension | Journal of Cell Biology | Rockefeller University Press (rupress.org) 29 Mechanical signal transduction amplifies the myosin recruitment Experimental application of tension by aspiration recruits myosin One piece of evidence that indicates myosin-induced tension at junctions recruits myosin (a positive feedback loop) Myosin II Dynamics Are Regulated by Tension in Intercalating Cells – ScienceDirect 30 Mechanical signal transduction amplifies the myosin recruitment Experimental application of tension by aspiration recruits myosin One piece of evidence that indicates myosin-induced tension at junctions recruits myosin (a positive feedback loop) How are the canonical signaling pathway and positive feedback loop connected to transcription factors of the anterior-posterior patterning system? Myosin II Dynamics Are Regulated by Tension in Intercalating Cells – ScienceDirect 31 Transmembrane receptors of the Toll family are expressed in distinct striped patterns downstream of the anterior-posterior patterning system Heterophilic interactions between Toll receptors occur in a planar polarized pattern and recruit myosin A positional Toll receptor code directs convergent extension in Drosophila | Nature 32 Transcription factors of the anterior posterior patterning system Toll receptor code Group discussion: Canonical myosin activation pathway What questions remain to be addressed to understand the mechanism of germband extension? Localized myosin activity T1T2 cell junction transition Cell intercalation and tissue elongation 33 In addition to the deployment of molecular machinery to change cell shape or cell-cell interactions, changes to cell numbers also drive tissue morphogenesis. 34 Global Effect Allometry: Differential growth of tissues or tissue regions shapes the body plan Figure 38.21 (macmillanhighered.com) 35 Local Effect Oriented cell division coupled with tissue elongation During zebrafish epiboly At the posterior tip of the Drosophila germband Oriented cell divisions in the extending germband of Drosophila | Development | The Company of Biologists Oriented cell division in vertebrate embryogenesis – ScienceDirect 36 Global Effect Apoptosis can sculpt tissues by removing cells 37 Local Effect The basal extrusion of an apoptotic cell can re-shape neighbouring cells (forming joints of the Drosophila leg) Death drags down the neighbourhood | Nature 38 In addition to driving forces, it is important to consider the mechanical properties of the materials the forces are acting on 39 In addition to driving forces, it is important to consider the mechanical properties of the materials the forces are acting on How do cells and tissues respond to forces? Are shape changes allowed or resisted? Are changes temporary or permanent? 40 In addition to driving forces, it is important to consider the mechanical properties of the materials the forces are acting on How do cells and tissues respond to forces? Are shape changes allowed or resisted? Are changes temporary or permanent? Are the basal domains of the brown cells able to expand? Is the neighbouring tissue allowed to spread? Apical pulling forces 41 Concepts from rheology: the study of the deformation of materials 1. Are shape changes allowed or resisted? Tensile force: A pulling force (could tear a tissue apart) Compressive force: A pushing force (could compact a tissue) Stress (σ): A measure of the tension/compression exerted within a material (force per unit area): σ = F/A Strain (ε): A measure of a material’s deformation from a starting shape: ε = (L – L0)/L0 Young’s modulus (E): The stress-strain relationship of a material: E=σ/ε (low value for a deformable material) Tensile Forces and Mechanotransduction at Cell–Cell Junctions – ScienceDirect 42 Concepts from rheology: the study of the deformation of materials 1. Are shape changes allowed or resisted? Tensile force: A pulling force (could tear a tissue apart) Compressive force: A pushing force (could compact a tissue) Stress (σ): A measure of the tension/compression exerted within a material (force per unit area): σ = F/A Young’s modulus (E): The stress-strain relationship of a material: E=σ/ε (low value for a deformable material) Tensile Forces and Mechanotransduction at Cell–Cell Junctions – ScienceDirect Concepts from rheology: the study of the deformation of materials 1. Are shape changes allowed or resisted? Tensile force: A pulling force (could tear a tissue apart) Compressive force: A pushing force (could compact a tissue) Stress (σ): A measure of the tension/compression exerted within a material (force per unit area): σ = F/A Strain (ε): A measure of a material’s deformation from a starting shape: ε = (L – L0)/L0 Young’s modulus (E): The stress-strain relationship of a material: E=σ/ε (low value for a deformable material) Tensile Forces and Mechanotransduction at Cell–Cell Junctions – ScienceDirect Concepts from rheology: the study of the deformation of materials 1. Are shape changes allowed or resisted? Tensile force: A pulling force (could tear a tissue apart) Compressive force: A pushing force (could compact a tissue) Stress (σ): A measure of the tension/compression exerted within a material (force per unit area): σ = F/A Strain (ε): A measure of a material’s deformation from a starting shape: ε = (L – L0)/L0 Young’s modulus (E): The stress-strain relationship of a material: E=σ/ε (low value for a deformable material) Tensile Forces and Mechanotransduction at Cell–Cell Junctions – ScienceDirect Concepts from rheology: the study of the deformation of materials 1. Are shape changes allowed or resisted? Tensile force: A pulling force (could tear a tissue apart) Compressive force: A pushing force (could compact a tissue) Stress (σ): A measure of the tension/compression exerted within a material (force per unit area): σ = F/A Strain (ε): A measure of a material’s deformation from a starting shape: ε = (L – L0)/L0 Young’s modulus (E): The stress-strain relationship of a material: E=σ/ε (low value for a deformable material) Is the neighbouring tissue allowed to spread? Apical pulling forces Tensile Forces and Mechanotransduction at Cell–Cell Junctions – ScienceDirect (compare the grey tissue with the blue box) Actin-based force generation and cell adhesion in tissue morphogenesis – ScienceDirect46 Concepts from rheology: the study of the deformation of materials 2. Are shape changes temporary or permanent? Elastic material: Elastic materials have a reversible and linear relationship between stress and strain. *Rubbers provide good examples of elastic materials. Viscoelastic fluid: A viscoelastic fluid presents solid-like behaviours at short time scales but behaves as a fluid at long time-scales, meaning that it will flow in response to stress. *Epithelia are typically viscoelastic fluids. Deformation can be temporary for a force applied over a short time scale Deformation can be permanent for a force applied over a long time scale Tensile Forces and Mechanotransduction at Cell–Cell Junctions – ScienceDirect 47 Concepts from rheology: the study of the deformation of materials 2. Are shape changes temporary or permanent? Elastic material: Elastic materials have a reversible and linear relationship between stress and strain. *Rubbers provide good examples of elastic materials. Viscoelastic fluid: A viscoelastic fluid acts as an elastic material at short time scales but behaves as a fluid at long time-scales, meaning that it will flow in response to stress. *Epithelia are typically viscoelastic fluids. Deformation temporary for a force applied over a short time scale Deformation permanent for a force applied over a long time scale Tensile Forces and Mechanotransduction at Cell–Cell Junctions – ScienceDirect 48 Epithelial behavior as a viscoelastic fluid Tensile force applied Tensile force removed No loss/gain of cellular/molecular interactions over short time scale (pre-existing configurations are deformed) Tensile Forces and Mechanotransduction at Cell–Cell Junctions – ScienceDirect 49 Epithelial behavior as a viscoelastic fluid Tensile force applied Quick removal of force Tensile force removed Deformation Reversed Tensile Forces and Mechanotransduction at Cell–Cell Junctions – ScienceDirect 50 Epithelial behavior as a viscoelastic fluid Tensile force applied Persistence of force Tensile force removed Cells and molecular networks dissipate stress by changing their organization Tensile Forces and Mechanotransduction at Cell–Cell Junctions – ScienceDirect 51 Epithelial behavior as a viscoelastic fluid Tensile force applied Persistence of force Tensile force removed Deformations Persist Tensile Forces and Mechanotransduction at Cell–Cell Junctions – ScienceDirect 52 Short break Advice about reading research articles Q&A session 53 How to engage scientific research articles Not casual reading  Active thinking is required Think about the questions raised… From the Introduction, identify the general questions being asked. – Why are they important? Through the Results, consider each experiment individually. – How do rationales (specific questions) fit with the general questions? – Is each experimental result clear and interpreted properly? From the Discussion, critically evaluate overall conclusions. -Have the general questions been addressed by the experimental results? -What remains to be investigated? Compare with topics discussed in class. Critically evaluate the data and the interpretations… Do you understand the methods, and are they appropriate for the investigating the questions?  search for methods fundamentals on-line  review the Monday lecture before the Wednesday lecture covering the article, and ask classmates Are the interpretations supported by the data?  interpret the data yourself – look for specific elements of the data – compare controls and experiments with only one difference – only assess the data in one figure (or figure panel) and ask whether this data supports a specific conclusion From all of the specific conclusions, do you agree with the overall model? Are there alternates? Critically evaluate the data and the interpretations… Do you understand the methods, and are they appropriate for the investigating the questions?  search for methods fundamentals on-line  review the Monday lecture before the Wednesday lecture covering the article, and ask classmates Are the interpretations supported by the data? Are additional tests or controls needed?  interpret the data yourself – look for specific elements of the data – compare controls and experiments with only one difference – only assess the data in one figure (or figure panel) and ask whether this data supports a specific conclusion From all of the specific conclusions, do you agree with the overall model? Are there alternates? Critically evaluate the data and the interpretations… Do you understand the methods, and are they appropriate for the investigating the questions?  search for methods fundamentals on-line  review the Monday lecture before the Wednesday lecture covering the article, and ask classmates Are the interpretations supported by the data? Are additional tests or controls needed?  interpret the data yourself – look for specific elements of the data – compare controls and experiments with only one difference – only assess the data in one figure (or figure panel) and ask whether this data supports a specific conclusion From all of the specific conclusions, do you agree with the overall model? Are there alternates? The pre-class quizzes test whether you are reading the assigned research articles in detail. They are “open book”, and are intended to be completed while you read the articles. Your understanding of the research articles needs to go beyond what is tested in the quizzes. The key elements that you need to understand are presented in the lectures, but a fuller understanding requires that you also critically read the research articles

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