Cells to Tissues

Peter TakizawaDepartment of Cell Biology

From one cell to ensembles of cells.

1. Been looking at basic properties of cells -> processes that most cells share.2. Start looking at how cells work together in tissues and organs to produce physiological effect.3. How cells adhere to each other.4. Communication between cells.5. How cells interact with extracellular matrix.

•Adhesion between cells

•Signaling between neighboring cells

•Cell adhesion to extracellular matrix

Cell to Cell Adhesion

Interactions between neighboring cells and between cells and ECM hold tissues together.

Adhering junctions

Desmosomes

Gap junctions

Integrins

1. Critical for cells to work in groups is maintaining connections between themselves.2. Several complexes hold cells together in tissues and organs.

2.1. Adhering junctions -> found in most tissues and cells.2.2. Desmosomes -> found primarily in cells subject to stress.2.3. Gap junctions -> communication between neighboring cells.

3. Cells form attachments to ECM.3.1. Set of fibers and structural molecules.3.2. Provide structural and metabolic support.3.3. Integrins.

4. Common themes.4.1. Connected to cytoskeleton.4.2. Adhesion molecules clustered to increase strength of interaction.4.3. Signaling platforms.

Cadherins are the adhesion molecule of adhering junctions and desmosomes.

1. Cadherins protein that mediates adhesion between cells in adhering junctions and desmosomes.2. Most single transmembrane proteins.3. Contain cadherin domains -> multiple copies. (6)

3.1. Number may determine space between cells.4. Interaction between cadherins occurs through most N-terminal domain.5. Cadherins localize to periphery of cells and between neighboring cells.6. Cadherins cluster where cells form initial attachments.7. Cadherins are adhesion molecule for adhering junctions and desmosomes.

Cadherins are the adhesion molecule of adhering junctions and desmosomes.

1. Cadherins protein that mediates adhesion between cells in adhering junctions and desmosomes.2. Most single transmembrane proteins.3. Contain cadherin domains -> multiple copies. (6)

3.1. Number may determine space between cells.4. Interaction between cadherins occurs through most N-terminal domain.5. Cadherins localize to periphery of cells and between neighboring cells.6. Cadherins cluster where cells form initial attachments.7. Cadherins are adhesion molecule for adhering junctions and desmosomes.

Cadherins are the adhesion molecule of adhering junctions and desmosomes.

1. Cadherins protein that mediates adhesion between cells in adhering junctions and desmosomes.2. Most single transmembrane proteins.3. Contain cadherin domains -> multiple copies. (6)

3.1. Number may determine space between cells.4. Interaction between cadherins occurs through most N-terminal domain.5. Cadherins localize to periphery of cells and between neighboring cells.6. Cadherins cluster where cells form initial attachments.7. Cadherins are adhesion molecule for adhering junctions and desmosomes.

Cadherins comprise a large family of proteins.

Desmosomal cadherin

E-cadherinN-cadherin

VE-cadherin

Protocadherin

1. Cadherins large family of proteins.1.1. All have basic cadherin domain with variable number of repeats.1.2. Most pass membrane once but some multiple times.

2. Founding member most common.2.1. Contains several tissue specific types.2.2. E -> epithelia.2.3. N -> neural tissue.2.4. VE -> endothelial cells.

3. Homotypic interaction.3.1. Cadherins of one group preferentially associated with members of same group.3.2. Important for development of tissues.

Cells can be sorted by types and expression level of cadherins.

N-cadherinE-cadherin

1. Example of how expression of different cadherins can separate cells.1.1. Mix cells expressing either E or N cadherin.1.2. Cells segregate from each other and cluster in groups that express same cadherin.

2. Cells also segregate if express different amount of cadherin.

Cells can be sorted by types and expression level of cadherins.

Low expressionHigh expression

1. Example of how expression of different cadherins can separate cells.1.1. Mix cells expressing either E or N cadherin.1.2. Cells segregate from each other and cluster in groups that express same cadherin.

2. Cells also segregate if express different amount of cadherin.

Changes in expression of cadherins leads to development of different tissues.

E-cadherin

N-cadherin

1. Differential expression of cadherins important for separating different tissues during development.2. Neural tube that will develop into ..... separates from ectoderm -> epithelia.3. Cells destined to become neural tube express N-cadherin to dissociate from ectoderm and start to cluster together.

Cadherin interactions are dependent on calcium.

1. Interaction between cadherins calcium sensitive.1.1. In absence of calcium cadherin domains folded over preventing interaction.1.2. Calcium causes cadherin domains to extend lengthening cadherin molecule.

2. Calcium opens terminal domain to allow interaction.

Clustering of cadherins increases strength of interactions between cells.

1. Interaction between individual cadherins is weak.2. Strength comes from clustering of cadherins in common domain.3. Similar to velcro.

Clustering of cadherins increases strength of interactions between cells.

1. Interaction between individual cadherins is weak.2. Strength comes from clustering of cadherins in common domain.3. Similar to velcro.

Links to cytoskeleton cluster cadherins in desmosomes and adhering junctions.

actin

intermediatefilaments

1. Electron micrographs show cadherins clustered in domains.2. Attachment to underlying cytoskeleton.3. Remove cytoskeleton or linkage causes cadherins to diffuse apart and weaken interaction.

Catenins link cadherins to actin filaments in adhering junctions.

β-catenin

α-catenin

1. Proteins in membranes diffuse rapidly due to thermal energy.2. Need something to hold them.3. Adhering junctions linked to actin filaments.

3.1. Prevents diffusion of cadherins.4. Don’t bind directly to actin but linked through set of proteins.

4.1. Catenins bind c-tail of cadherins.4.2. Set of other proteins links catenins to actin.

In desmosomes, cadherins are linked to intermediate filaments.

Desmoplakin

Plakophilin

Plakoglobin

1. Cadherins in desmosomes linked to intermediate filaments via set or proteins.2. Linkage to IFs means desmosomes involved in generating mechanical strength between cells and allowing for stretching.3. Desmosomes prominent in cells and tissues subjected to mechanical stress -> skin.

Signaling between neighboring cells

Cell contacts regulate cell division and coordinate activities between cells.

Cell CycleCell Cycle

Biochemical Pathways

Biochemical Pathways

Cell to cell contacts not only play a critical role in holding cells together in a tissue but intercellular connections also function as means of communication between cells. Cells in common tissues coordinate many of their activities (cell division, metabolism, morphology) and the intercellular connections mediate this coordination. For example, many cells will divide in culture dishes until they make contact with another cell. The cell connections cause both cells to exit from the cell cycle and remain quiescent.

Beta-catenin in adhering junctions is a transcription factor activated by Wnt signaling.

Wnt signaling

β-catenin

1. Beta-catenin links cadherins to actin.2. Beta-catenin functions as transcription activator -> cell proliferation3. Two pools.

3.1. Bound to cadherin -> can’t activate transcription.3.2. Cytosolic pool -> phosphorylated by complex of proteins that target it for degradation.

4. Wnt signaling inhibits complex, stabilizing beta-catenin -> activation of transcription.4.1. Wnt important during development -> cell proliferation, morphogenesis, cell-fate.4.2. Beta-catenin activates transcription of myc.

5. In absence of Wnt, catenin degraded or in complex with cadherin.6. Disruption of cadherin, releases catenin -> some escapes degradation -> transcription activation.7. Relationship between cell adhesion and cell proliferation.8.

Neighboring cells communicate directly through gap junctions.

Gap junction

Plasma membrane

1. Cells communicate directly through gap junctions.2. Collection of protein pores in plasma membrane that interconnect cells.

2.1. Close apposition of PM.2.2. Large cluster of pores.

3. Cells increase decrease size of gap junctions via exocytosis and endocytosis.

Gap junctions allow diffusion of small molecules between neighboring cells.

1. Pores of gap junctions size restrictive pores.2. Molecules smaller than 1000 Da can freely diffuse through pores.3. Ions and most metabolites can pass.4. Cells with large gap junctions have cytoplasm with common ionic composition and share metabolites and nutrients.5. Allows cells to relay signals -> activation of cell can be passed to neighboring cells.

5.1. Cardiac muscle.

Connexins are transmembrane proteins that form ~1.5 nm pores between cells.

1.5 nm pore

1. Connexins proteins that make up pores.2. 6 connexins assemble in membrane of one cell to form pore.3. Connexins in neighboring cells interact to form channels between cells.4. Family of proteins.

4.1. Mutations in connexin 26 cause most common form of congenital deafness.

Calcium causes connexin pores to shrink and prevent loss of material in damaged cells.

Nutrients

Ca2+

1. Problem with interconnected cells -> damage to one cell could affect all cells in tissue.2. Breaks in plasma membrane allow contents to leak.

2.1. Contents from surrounding undamaged cells could leak through gap junctions.3. Calcium outside cell enters damaged cell.

3.1. High extracellular calcium, low intracellular.3.2. Connexins calcium sensitive.3.3. Close when exposed to calcium.

Calcium causes connexin pores to shrink and prevent loss of material in damaged cells.

Ca2+ free 0.5 mM CaCl2

Pore size (nm) 1.3 ± 0.3 0.5 ± 0.3

Müller et al EMBOJ 21 3598-3607

1. Problem with interconnected cells -> damage to one cell could affect all cells in tissue.2. Breaks in plasma membrane allow contents to leak.

2.1. Contents from surrounding undamaged cells could leak through gap junctions.3. Calcium outside cell enters damaged cell.

3.1. High extracellular calcium, low intracellular.3.2. Connexins calcium sensitive.3.3. Close when exposed to calcium.

Extracellular matrix

Extracellular matrix contains protein fibers, proteoglycans and hyaluronon.

Collagen

1. Composition.1.1. Set of fibrous proteins, proteoglycans (proteins with sugars) and other molecules.

2. Functions.2.1. Mechanical strength -> resist stretching and compression.2.2. Metabolic support.

2.2.1. Retain and control diffusion of nutrients.2.2.2. Control diffusion of signaling molecules.

2.3. Cell behavior.2.3.1. Regulates cell growth and differentiation.2.3.2. Motility.

3. Components often replace cells during disease.

Collagen is the main structural component of extracellular matrix.

1. Collagen has long helical domain.1.1. Coiled coil forms trimers.1.2. Wrap around each other.1.3. Mechanical strength.

2. Individual collagens crosslinked out side cells3. Individual collagens polymerize into fibers outside cell.4. Produced by fibroblasts.

4.1. Produce most components of ECM.

Collagen is the main structural component of extracellular matrix.

1. Collagen has long helical domain.1.1. Coiled coil forms trimers.1.2. Wrap around each other.1.3. Mechanical strength.

2. Individual collagens crosslinked out side cells3. Individual collagens polymerize into fibers outside cell.4. Produced by fibroblasts.

4.1. Produce most components of ECM.

Elastin allows for stretching and recoil of extracellular matrix.

1. Elastin proteins are crosslinked to one another to form an interconnected network of proteins. 2. Elastin is unstructured and the protein adopts coils with random orientations.

2.1. Elastin is a disorganized mesh of crosslinked protein. 2.2. When a force pulls on elastic fibers, elastin begins to lengthen and become more structured with proteins running in parallel arrays.

3. The reason that elastin recoils is based on thermodynamics.3.1. Systems become disordered unless energy is put into the system. 3.2. External force provides energy to order the elastin proteins. 3.3. Once that force is removed, the elastin wants to return to the disordered state and in the process it releases energy that generates a recoiling force. Rubber bands work the same way.

Proteoglycans are proteins with long side sugar side chains.

1. Proteins with many sugar side chains.2. Sugars are negatively charged.

1. Attract cations and water.2. Take up space.

3. Interact with signaling molecules to control diffusion.1. Prevent movement into blood stream.2. Keep local high concentration.

4. O-linked with repeated disaccharide.1. Glycosaminoglycan (GAGs)

Proteoglycans are proteins with long side sugar side chains.

1. Proteins with many sugar side chains.2. Sugars are negatively charged.

1. Attract cations and water.2. Take up space.

3. Interact with signaling molecules to control diffusion.1. Prevent movement into blood stream.2. Keep local high concentration.

4. O-linked with repeated disaccharide.1. Glycosaminoglycan (GAGs)

Hyaluronan resists compression and sequesters water.

24,988 disaccharides

1. Hyaluronan is another glycosaminoglycan and is the primary compression-resisting component of connective tissue. 2. Hyaluronan does not contain protein.

2.1. Long polymer of repeating disaccharides. 2.2. Contain up to 25000 repeats.2.3. Reach a length of 20 µm, the size of an average cell.

3. Hyaluronan lacks the structure of most proteins and contains many regions of that form random, flexible coils. 3.1.Sugars in hyaluronan are negatively charged and repel each other. 3.2. Generates a lot of space within hyaluronan and allows it to occupy an incredibly large volume. 3.3. Retains water. 3.4. Functions as that water filled bottle to resist compression.

Hyaluronan resists compression and sequesters water.

1. Hyaluronan is another glycosaminoglycan and is the primary compression-resisting component of connective tissue. 2. Hyaluronan does not contain protein.

2.1. Long polymer of repeating disaccharides. 2.2. Contain up to 25000 repeats.2.3. Reach a length of 20 µm, the size of an average cell.

3. Hyaluronan lacks the structure of most proteins and contains many regions of that form random, flexible coils. 3.1.Sugars in hyaluronan are negatively charged and repel each other. 3.2. Generates a lot of space within hyaluronan and allows it to occupy an incredibly large volume. 3.3. Retains water. 3.4. Functions as that water filled bottle to resist compression.

Fibronectin links cells to extracellular matrix.

1. Link cell to extracellular matrix.1. Contain domain that binds components of ECM (collagen).2. Contain domain that binds receptors in cell membrane.

2. Dimer held together by disulfides.3. Form fibers under tension.4. Forms small aggregates at edge of cell.5. Fibers where receptors are attached to actin cytoskeleton -> tension.

Fibronectin links cells to extracellular matrix.

Fibronectin

1. Link cell to extracellular matrix.1. Contain domain that binds components of ECM (collagen).2. Contain domain that binds receptors in cell membrane.

2. Dimer held together by disulfides.3. Form fibers under tension.4. Forms small aggregates at edge of cell.5. Fibers where receptors are attached to actin cytoskeleton -> tension.

Integrins are cell surface receptors that bind components of extracellular matrix.

1. Adhesion to ECM mediated by integrins.1.1. Single transmembrane proteins.1.2. Form heterodimers.

2. Interact with fibers in ECM.2.1. Fibronectin.2.2. Laminin.

3. Linked internally to cytoskeleton.3.1. IFs -> hemidesmosome.

3.1.1. Mainly in epidermis.3.1.2. Dystonin and plectin.

3.2. Linked to actin in most cells.4. Clustering increases strength of interaction.

Integrins are heterodimers that are linked to the cytoskeleton.

α-subunit β-subunit

1. Integrins composed of alpha and beta subunits.1.1. 18 alphas.1.2. 8 betas.1.3. 24 combinations.1.4. Certain combinations ubiquitous and others tissue/cell specific.

2. Integrins linked to actin via talin that binds beta subunit.2.1. Talin helps assemble actin filaments.

Strength of interaction between integrins and extracellular matrix is regulated.

1. Strength of integrin interaction with ECM can be regulated by external and internal signals.2. Integrins in two conformations.

2.1. No ligand present.2.1.1. Beta next to alpha -> can’t interact with talin.

2.2. Bound to ligand.2.2.1. Integrins unfolded.2.2.2. Beta subunit moves away from alpha -> binds talin.

3. Outside-in.3.1. Presence of ligand in ECM unfolds integrins.3.2. Allows integrins to bind talin and assemble actin filaments at adhesion site.

4. Inside-out.4.1. Cells activate talin.4.2. Talin binds inactive integrins to separate integrins and convert to high affinity binding state.

5. Talin activated by G-protein and RTK pathways.6. Signals tell cells whether to tightly adhere to ECM.

Strength of interaction between integrins and extracellular matrix is regulated.

1. Strength of integrin interaction with ECM can be regulated by external and internal signals.2. Integrins in two conformations.

2.1. No ligand present.2.1.1. Beta next to alpha -> can’t interact with talin.

2.2. Bound to ligand.2.2.1. Integrins unfolded.2.2.2. Beta subunit moves away from alpha -> binds talin.

3. Outside-in.3.1. Presence of ligand in ECM unfolds integrins.3.2. Allows integrins to bind talin and assemble actin filaments at adhesion site.

4. Inside-out.4.1. Cells activate talin.4.2. Talin binds inactive integrins to separate integrins and convert to high affinity binding state.

5. Talin activated by G-protein and RTK pathways.6. Signals tell cells whether to tightly adhere to ECM.

Integrins cluster into focal adhesions that function as signaling platforms.

ActinFocal Adhesion

1. Integrins cluster into large macromolecular assemblies called focal adhesions.1.1. Contain many integrins.1.2. Linked to actin cytoskeleton.

2. Function as signaling platform.2.1. Relay state of ECM.2.2. How strongly cell is attached to ECM -> tension from actin filaments.

Integrins cluster into focal adhesions that function as signaling platforms.

1. Integrins cluster into large macromolecular assemblies called focal adhesions.1.1. Contain many integrins.1.2. Linked to actin cytoskeleton.

2. Function as signaling platform.2.1. Relay state of ECM.2.2. How strongly cell is attached to ECM -> tension from actin filaments.

Stiffness of ECM regulates cell survival and proliferation.

1. Composition of ECM affects cell behavior.2. Too soft prevents cells from forming strong adhesion.

2.1. Can’t spread2.2. Can’t generate tension.

3. Stiff matrix allow cells to generate strong adhesion and proliferate.4. Intermediate allows cell motility.