Learning Objectives

• Nature and Role of ECM Proteins: Understand ECM components including collagen.

• Collagen Types: Differentiate between various types of collagen.

• Integrin Conformations: Identify roles that different conformations of integrins play in mammalian cells.

• Adhesion Molecules: Focus on the steps and functions of adhesion molecules in phenomena like extravasation.

• Plasmodesmata vs Intercellular Adhesion: Differentiate their roles in plant and mammalian cells.

• Intermediate Filaments: Characterize and differentiate the functions and distributions of various intermediate filaments.

Selected Collagens - Table 20.5

Page 4: Type IV Collagen Structure

• Found in all Basal Laminae

• Characteristics: 400-nm long triple helical structures with 24 non-helical interruptions adding flexibility.

• Association and Network Formation: Flanked by globular domains for interaction; covalently cross-linked to form networks, attaching to laminin to create the basal lamina.

Perlecan (proteoglycan)

• Description: Major secreted proteoglycan ECM component within basal laminae.

• Structure: Large multidomain core protein with multiple polysaccharide side-chains.

• Function: Binds with various other molecules like nidogen and laminin to mediate cell-surface receptor interactions.

• 

Collagen Structure and Assembly

• Triple-Helical Structure: Assembles characterized by G - X - Y repeat motif where G is glycine (central), X is often proline, and Y is hydroxyproline.

• Fiber Formation: Microfibrils (300 nm) aggregate to form fibrils and further bundle into long fibers.

Type I Properties

• Fibrillar Molecules: Most abundant; largely present in connective tissues (Types I, II, III).

• Fibril-Associated Collagens: Maintain structural integrity by bridging fibrillar collagens with each other or to the ECM.

• Sheet-Forming Collagens: Type IV forms 2D networks crucial to basal laminae.

• Type I Collagen: Known for exceptional tensile strength, used heavily in bone reinforcement (30% protein, 70% mineral). Stronger than steel, gram for gram

Collagen Synthesis and Assembly

• Collagen Production: Occurs in the rough endoplasmic reticulum (ER), Golgi apparatus, and extracellular space.

• Procollagen to Collagen Molecule: Involves propeptide cleavage and lateral association leading to the assembly of collagen fibers.

Glycosaminoglycans (GAGs)

• Role in ECM: Proteoglycans crucial for adhesion, existing in secreted or membrane-attached forms.

• Classifications: Four types differentiated by disaccharide repeats and modifications, contributing to the structure of proteoglycans. Covalently linked to specialized polysaccharide side-chains (GAGs)

GAG Synthesis

• Core Syntheses: Proteoglycan cores synthesized in the ER; GAG chains assembled in the Golgi.

• Linkage Variations: GAGs may be O-linked (attached by the -OH moiety in Serine or Threonine R-groups) or N-linked (attached by the R-group of the asparagine residues).

Hyaluronan (Hyaluronic Acid, HA)

• Definition: A non-sulfated GAG, significant for ECM structure, made by membrane bound HA-synthase and secreted into the ECM. Forms the backbone of ECM proteoglycan aggregates.

• Characteristics: Forms hydrated spheres/gel; resists compressive forces, predominantly found in cartilage.

• 

Fibronectin

• Description: An important ECM component across vertebrates; composed of dimers of 2 similar polypeptides linked at C-terminus linked by disulfide bonds.

• Function: Attaches cells to ECM via binding to fibrillar collagen, proteoglycans, and adhesion receptors; contains specific binding domain (RGD) for integrin.

• Domains are Type I, II, or III. Arg-Gly-Asp domain binds Integrin

• 

Page 13: Elastic Fibers

• Structure: Composed of fibers with elastin cores; essential for tissues under mechanical strain or deformation like lungs.

• Components: Elastin is covalently cross-linked tropoelastin aggregates surrounded by collagen microfibrils. Elastin core of fibers.

  

Matrix Metalloproteases (MMPs)

• ECM Dynamics: The ECM is subject to remodeling and degradation primarily by zinc-dependent MMPs.

• Subgroups of MMPs: Include MMPs for remodeling and degradation, ADAMs for integrin degradation, and ADAMTSs for signaling functions.

• ADAM: Asidintegrin and metalloproteinases

Page 15: Integrins and Adhesion

• Dynamic Adhesion: Integrins connect the ECM and cytoskeleton, clustering into various adhesive structures.

  • focal adhesions in 2D culture

  • 3D adhesion in 3D cultures

  • Fibrillar adhesions

  • Podosomes (“foot bodies”)

Page 16: Integrin Binding Affinity States

• 3 Conformational States that correspond to binding affinity:

  • Bent Closed

  • Extended Closed

  • Extended Open

• Functionality: These states correlate with binding affinity of integrins.

• 

Signaling through Integrins

• Cytosolic Domains: Integral proteins can signal and connect to the cytoskeleton; molecules like talin modify phospholipids and allows association, leading to integrin activation and complex formation. Association in turn causes: activation, dimerization, complex building, integrin activation, recruitment and force

Fibroblasts

• Role: Major secretors of ECM; critical for maintaining ECM dynamics.

• Location: Present in corneal structures, including Bowman's layer, Descemet's membrane, and stroma.

Cell Motility in Immunity

• Importance: Motility vital for wound healing and immune responses; immune cells are recruited via extravasation to areas needing intervention.

• 

Plant Cell Walls

• Structure: Comprised of polysaccharides forming ECM-like structures, mainly cellulose, hemicellulose, and pectin. Cellulose is synthesized outside the cell membrane, while hemicellulose and pectin are ER synthesied

• Function: Resist turgor pressure (like air filling a balloon), produced externally to the cell membrane.

• 

Page 21: Plasmodesmata in Plants

• Description: Connect adjacent plant cells through structures that share an ER membrane; allow the transfer of molecules between cells.

• Cell walls means almost no CAMs

• Plants have plasmodesmata structures

  that connect adjacent cells

• Plasmodesmata share an ER membrane that lines the structure (the annulus) that is 30-60nm to >1μm in diameter

• A narrow tube (desmotubule) is formed with a regulated gap of 2-10 nm called the cytoplasmic sleeve

• Allows free transfer of small molecules or regulated transfer of large ones

Page 22: Intermediate Filaments

• Characteristics: 10 nm thick, stable structures with biochemically heterogenous group, great tensile strength, no inttrinsic polarity, no motore proteins associated, much more stable than the other 2 filaments, and are not found in all eukaryotic cells.

• Major Classes of Intermediate Filaments

  Class	Protein	Distribution	Proposed Function
  I	Acidic keratins	Epithelial cells	Tissue strength and integrity
  II	Basic keratins	Epithelial cells	Tissue strength and integrity
  III	Desmin, GFAP, vimentin	Muscle, glial cells, mesenchymal cells	Sarcomere organization, integrity
  IV	Neurofilaments (NFL, NFM, and NFH)	Neurons	Axon organization
  V	Lamins	Nucleus	Nuclear structure and organization

• Structure of Intermediate Filaments

  • Conserved dimerized 310 aa a helical rod domain

  • Basic unit = dimer, dimers form anti parallel tetrads, tetrads unite to form protofilaments, 4 protofilaments unite to form a protofibril, 4 protofibril unite to form an IF

• 

• Variability in Intermediate Filaments

  • Structural Differences: Variances in non-helical domains complicate formation; involvement of non-nucleating, capping, sequestering, or severing proteins.

• Polarity of Intermediate Filaments

  • Classes of Keratins make up:

    • Class I and II: in epithelia

    • Class III in mesoderm

    • Class IV in neurofilaments

    • Class V in the nucleus of all our cells

• 

• Stability of Intermediate Filaments

  • Dynamic Structures: Incorporation of free units into stable structures; keratin in epithelial appendages provides mechanical strength.

  • Free IF units can be incorporated into existing structures.

  • Keratin dimer units are acidic/basic pairs. Keratins make hair and nails hard. Cytokeratins strengthen connection to make a tight skin/dermal layer.

Lamins in the Nucleus

• Function: Support the nuclear envelope and maintain chromatin structure; specific classes (A/C and B1/B2) with distinct associations and functions.

• Lamins are the meshwork on the inside of our nuclei, connecting the membrane and chromatin

  • Lamin A provides rigidty

  • Lamin B associated with receptors and helps attach the nucleus to the cytoskeleton through SUN and KASH domain containing proteins

• 

Intermediate Filament-Associated Proteins (IFAPs)

• Role: Support the connections between intermediate filaments and other cytoskeletal components, enhancing cellular maintenance and integrity.

• Co purify with IFs

• IFAPs include plakin family members. Plakins link IF to the desmosomes and hemidesmosomes. Other plakins connect IFs to microfilaments and microtubules.