Dr Saeb Aliwaini

• Often the cell does not behave as in vivo because:
1- loss of heterogeneity and three-dimensional architecture.
2- Hormonal and nutritional stimuli are not the same!
Therefore cells commonly favor the spreading, migration, and proliferation of unspecialized cells, rather than the expression of differentiated functions.
Dr Aliwaini

• Nature of the substrate
• Degree of contact with other cells
• Constitution of the medium
• Constitution of the gas phase
• The incubation temperature
Dr Aliwaini

• Solid tissues grow as adherent minelayers, and unless they have transformed and become anchorage independent
• What is required for proliferation?
- Glass with a slight net negative charge and some plastics, such as polystyrene treated with strong acid, a plasma discharge, or highenergy ionizing radiation
- Spreading may be preceded by the cells’ secretion of extra cellular matrix proteins and proteoglycans.
- To bind to the matrix via specific receptors
Dr Aliwaini

• 2 nd hand glass or plastic, are they Ok to use ?
• Fibronectin or collagen, or derivatives such as gelatin can also help cells to ------------------- and ---------------------
• Epithelial cells require cell–cell adhesion for optimum survival and growth, and consequently they tend to grow in patches.
• while fibroplasts have other features.
Dr Aliwaini

Dr Aliwaini

Dr Aliwaini

Dr Aliwaini

• To understand this you need a short revision about tissues in vivo.
Dr Aliwaini

• Connective tissue
• Epithelial tissue
• Muscles
• Nerves
• In connective tissues, extracellular matrix is plentiful and carries the mechanical load.
• We discuss connective tissues fist
Dr Aliwaini

• Many types of CT.
• In all of these tissues, the tensile strength—whether great or small— is chiefly provided by a fibrous protein: collagen.
• The various types of connective tissues owe their specific characters to the type of collagen that they contain, to its quantity, and, most importantly, to the other molecules that are interweave with it in varying proportions (elastin, as well as a host of specialized polysaccharide molecules)
Dr Aliwaini

• Mammals have about 20 different collagen genes.
• They constitute 25% of the total protein mass in a mammal
• long, stiff, triple-stranded helical structure, in which three collagen polypeptide chains are wound around one another in a ropelike super helix.
Dr Aliwaini

Dr Aliwaini
The cell in the photograph is a firoblast, which secretes the collagen as well as other extracellular matrix components
Other collagen molecules decorate the surface of collagen fibrils and link the fibrils to one another and to other components in the extracellular matrix

• In skin, tendon, and many other connective tissues they are called firoblasts, in bone they are called osteoblasts.
Dr Aliwaini

• The cells secret collagen molecules in a precursor form, called procollagen, with additional peptides at each end that obstruct assembly into collagen fibrils.
• Extracellular procollagen proteinases—cut off these terminal domains to allow assembly only after the molecules have emerged into the extracellular space.
Dr Aliwaini

• Cells in tissues have to be able to degrade matrix as well as make it.
• Matrix proteases ( arthritis and cancer)
• cells organize the collagen that they secrete
• integrins couple the matrix outside a cell to the cytoskeleton inside it
Dr Aliwaini

• Cells do not attach well to bare collagen.
• Another extracellular matrix protein , fibronectin, provides a linkage:
• one part of the fibronectin molecule binds to collagen, while another part forms an attachment site for a cell.
• Cell attaches itself to this specific site in fibronectin by means of a receptor protein, called an integrin, which spans the cell’s plasma membrane.
Dr Aliwaini

Dr Aliwaini

• Integrins do more than passively transmit stress:
They also react to stress—and to chemical signals from inside and outside the cell that direct them to maintain their attachment to other molecules or to let go.
Integrins form and break attachments, for example, as a cell crawls through a tissue, grabbing hold of the matrix at its front end and releasing its grip at the rear.
Dr Aliwaini

Dr Aliwaini
Integrins perform these functions by undergoing remarkable conformational
.
changes
These conformational changes in integrins are used to transmit chemical as well as mechanical signals
.
across the cell membrane

Binding to a molecule on one side of the membrane causes the integrin molecule to stretch out into an extended, activated state so that it can then latch onto another molecule on the opposite side—an effect that operates in both directions across the membrane
Dr Aliwaini

• In this way, the external attachments that a cell makes help to regulate whether it lives or dies, and—if it does survive— whether it grows, divides, or differentiates.
• Humans make at least 24 different kinds of integrins
•
• leucocyte adhesion deficiency
Dr Aliwaini

Dr Aliwaini

• Gels of polysaccharide and protein Fill spaces and resist compression
• Complementary function for proteoglycans, extracellular proteins linked to a special class of complex negatively charged polysaccharides, the glycosaminoglycans (GAGs)
Dr Aliwaini

Dr Aliwaini

• Proteoglycans are extremely diverse in size, shape, and chemistry.
• Typically, many GAG chains are attached to a single core protein, which may in turn be linked at one end to another
GAG, creating an enormous macromolecule resembling a bottlebrush, with a molecular weight in the millions of daltons.
Dr Aliwaini

Dr Aliwaini

Dr Aliwaini

Proteoglycan ( such as aggrecan)
Any protein with one or more covalently attached glycosaminoglycan chains.
Hyaluronan
A glycosaminoglycan defined by the disaccharide unit (GlcNAcβ1–4GlcAβ 1–3) n that is neither sulfated nor covalently linked to protein. It is referred to in older literature as hyaluronic acid.
Dr Aliwaini

• Bone have collagen plus calcium phosphate crystals.
• jellylike substance in the interior of the eye consists almost entirely of one particular type of GAG, plus water, with only a small amount of collagen
• In general, GAGs are strongly hydrophilic and tend to adopt highly extended conformations, which occupy a huge volume relative to their mass .
Dr Aliwaini

• GAGs have multiple negative charges attracting a cloud of cations, such as Na+, that are osmotically active, causing large amounts of water to be sucked into the matrix
• Proteoglycans, can bind secreted growth factors and other proteins that serve as signals for cells
• They can block, encourage, or guide cell migration through the matrix
Dr Aliwaini

Dr Aliwaini

Focal Adhesions Anchoring Cells to Their Substratum
• At first, the cell has a rounded morphology
• it sends out projections that form increasingly stable attachments
Dr Aliwaini

• When fibroblasts or epithelial cells spread onto the bottom of a culture dish, the lower surface of the cell is not pressed uniformly against the substratum. Instead, the cell is anchored to the surface of the dish only at scattered, discrete sites, called focal adhesions.
• Focal adhesions are dynamic structures that can be rapidly disassembled if the adherent cell is stimulated to move or enter mitosis.
Dr Aliwaini

Dr Aliwaini

Transmembrane proteoglycans also interact with matrix constituents such as other proteoglycans or collagen act as lowaffinity growth factor receptors.
36 Dr Aliwaini

Dr Aliwaini

• There are more than 200 visibly different cell types in the body .
• The majority of these are organized into epithelia in which the cells are joined together, side to side, to form multicellular sheets
Dr Aliwaini

• protective barrier
• Some secrete specialized products such as hormones, milk, or tears; others, such as the epithelium lining the gut, absorb nutrients; yet others detect signals, such as light, sensed by the layer of photoreceptors in the retina of the eye, or sound, sensed by the epithelium containing the auditory hair cells in the ear
Dr Aliwaini

• Epithelial sheets are polarized and rest on a Basal lamina:
• The apical surface and the the basal surface
Supporting the basal surface of the epithelium is a thin tough sheet of extracellular matrix called the, basal lamina
Composed of a specialized type of collagen (Type IV collagen) and various other macromolecules. These include a
laminin protein
Dr Aliwaini

Basement membranes contain two network-forming molecules, collagen IV
(pink) and laminin (green), which is indicated by the thickened cross-shaped molecules. The collagen and laminin networks are connected by entactin molecules (purple).
Dr Aliwaini

• laminin, provides adhesive sites for integrin molecules in the plasma membranes of the epithelial cells, and thus serves a linking role like that of fibronectin in connective tissues.
• polarized organization: Each has a top and a bottom, with different properties
Dr Aliwaini

The tightest attachment between a cell and its extracellular matrix is seen at the basal surface of epithelial cells
Hemidesmosome consists of the protein keratin ( intermediate filaments)
.
Dr Aliwaini

• The transmembrane protein BP180 has a large extracellular domain (ECD) which consists of 15 interrupted collagenous subdomains ( Giudice et al,
1992 ; Hopkinson et al, 1992 ). It serves as a cell-surface receptor, contributing to the maintenance of dermo-epidermal cohesion by binding most likely to the extracellular matrix component laminin 5 ( Giudice et al, 1992 ; Hopkinson et al,
1992 ; Borradori et al, 1999 ; Nievers et al, 2000 ; Nishiyama et al, 2000 ). Furthermore, BP180 is probably involved in epidermal differentiation by shedding and facilitating detachment of keratinocytes from the basal cell layer ( Giudice et al,
1992 ; Hopkinson et al, 1992 ; Borradori et al, 1999 ; Nievers et al, 2000 ; Nishiyama et al, 2000 ). Mutations of the BP180 gene (COL17A1) may cause a clinical variant of non-Herlitz junctional epidermolysis bullosa, a heritable disorder characterized by skin fragility and blistering ( McGrath et al, 1995 ; Pulkkinen et al, 1998 ).
Dr Aliwaini

• Selectins
1- E-selectin, present on endothelial cells
2- P-selectin, present on platelets and endothelial cells
3- L-selectin, present on leukocytes (white blood cells).
Selectins depends on Ca 2+ ions
We are taking a bout something like what?
Dr Aliwaini

• Are a large family of glycoproteins that mediate Ca2 + dependent cell–cell adhesion and transmit signals from the ECM to the cytoplasm.
• Cadherins typically join cells of similar type to one another and do so predominantly by binding to the same cadherin present on the surface of the neighboring cell.
• E-cadherin (epithelial),
• N-cadherin (neural),
• and P-cadherin (placental)
Dr Aliwaini

Dr Aliwaini

The classical cadherins are Ca2+- dependent and are involved primarily in interactions between homologous cells either via
- Adherens junctions (cadherins E, N, P, and VE)
Cadherins E, N, P, and VE connect to the actin cytoskeleton and has a signaling, as well as structural, role acting via α- and β-catenins, vinculin, and α-actinin
- Desmosomes (desmoglein, desmocollin)
48 Dr Aliwaini

• Adherens junctions
In an adheren junction, cells are held together by calciumdependent linkages formed between the extracellular domains of cadherin molecules that bridge the 30-nm gap between neighboring cells cytoplasmic domain of these cadherins is linked by catenins to a variety of cytoplasmic proteins, including actin filaments of the cytoskeleton.
Dr Aliwaini

Dr Aliwaini
Epithelial cells contain a continuous band of cadherin molecules, usually located near the apical surface just below the tight junction, that connects the lateral membranes of epithelial cells

• The cadherins of desmosomes have a different domain structure from the classical cadherins found in adherens junctions and are referred to as desmogleins and desmocollins
Dr Aliwaini

• A desmosome consists of proteinaceous adhesion plaques (15 – 20 nm thick) attached to the cytosolic face of the plasma membranes of adjacent cells and connected by transmembrane linker proteins.
• Plakoglobin is a major constituent of the plaques
• The transmembrane linker proteins, called desmoglein and
desmocollin, belong to the cadherin family
• provide strength and rigidity to the entire epithelial cell layer
Dr Aliwaini

Dr Aliwaini

Occludin and claudins They bind tightly to each other across the gap between adjacent cells helps establish cell polarity
- The main role of tight junctions is to seal the intercellular space so that any molecules traveling from the apical to basal surface, and vice versa, must pass through the cell in a regulated fashion
Dr Aliwaini

GAP JUNCTIONS MEDIATING INTERCELLULAR
COMMUNICATION
• Gap junctions are sites between animal cells that are specialized for intercellular communication
• They are composed entirely of an integral membrane protein called connexin.
• Connexins are organized into multisubunit complexes, called connexons,
Dr Aliwaini

Dr Aliwaini

• It varies for
• 1- mechanical junctions such as :
- The desmosomes and adherens junctions, which hold epithelial cells together.
- Tight junctions, which seal the space between cells such as between secretory cells in an acinus or duct or between endothelial cells in a blood vesse.
- 2- Transport : Gap junctions, which allow ions, nutrients, and small signaling molecules such as
Dr Aliwaini

• As epithelial cells differentiate in confluent cultures, they can form an increasing number of desmosomes and, if some morphological organization occurs, can form complete junctional complexes of adherens and tight junctions.
Dr Aliwaini

• If this happened; Epithelial cells will be more resistant to disaggregation, as they tend to have tighter junctional complexes
(desmosomes, adherens junctions, and tight junctions) holding them together.
• Whereas mesenchymal cells, which are more dependent on integrin–matrix interactions for intercellular bonding, are more easily dissociated
Dr Aliwaini

• Homophilic binding of cadherins and integrin receptor binding to matrix constituents are both dependent on divalent cations
Ca2+ and Mg2+. Hence trypsin EDTA, are often used to enhance disaggregation.
Dr Aliwaini

• Fibrocytes secrete type I collagen and fibronectin into the matrix).
• Epithelial cells produce laminin
• When you arrange combined culture : cell types are different,
(fibrocytes) and epidermis (keratinocytes), both cell types will contribute to the composition of the ECM, often producing a basal lamina
Dr Aliwaini

• ECM is comprised variously of collagen, laminin, fibronectin, hyaluronan, and proteoglycans such It can be prepared by mixing purified constituents, such as collagen and fibronectin, by using cells to generate
ECM and washing the producer cells off before reseeding with the cells under study .
• Commercially available matrices , such as Matrigel™ (Becton Dickinson) from the Engelbreth–Holm–Swarm (EHS) sarcoma, contain laminin fibron ectin , and proteoglycans , with laminin predominatig.
Dr Aliwaini

Next is cytoskeleton and cell proliferation
Dr Aliwaini