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Biology of Cultured Cells
Dr Saeb Aliwaini
THE CULTURE ENVIRONMENT
• 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
How does environment affect culture ?
• Nature of the substrate
• Degree of contact with other cells
• Constitution of the medium
• Constitution of the gas phase
• The incubation temperature
Dr Aliwaini
CELL ADHESION
• 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 high-energy 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
• 2nd 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
What cells grow in colonies and what don’t care ??
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Cell Adhesion Molecules
• To understand this you need a short revision about tissues in vivo.
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Tissues
• Connective tissue• Epithelial tissue• Muscles • Nerves • In connective tissues, extracellular matrix is plentiful and carries
the mechanical load.
• We discuss connective tissues fist
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• 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
Collagen
• 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
The cell in the photograph isa firoblast, which secretes the collagen as well as other extracellular matrixcomponents
Other collagen molecules decorate the surfaceof collagen fibrils and link the fibrils to one another and to other components in the extracellular matrix
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• In skin, tendon, and many other connective tissues they are called firoblasts, in bone they are called osteoblasts.
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• 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.
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• 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
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• 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.
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Integrins perform these functions by undergoing remarkable conformational changes.
These conformational changes in integrins are used to transmit chemicalas well as mechanical signals across the cell membrane.
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Binding to a molecule on one side of the membranecauses the integrin molecule to stretch out into an extended, activatedstate so that it can then latch onto another molecule on the oppositeside—an effect that operates in both directions across the membrane
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• 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
What else?
• 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
Dr Aliwaini
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.
Proteoglycan ( such as aggrecan)
Any protein with one or more covalently attached glycosaminoglycan chains.
It is not the same for all CT!
• 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
Focal adhesions
• When fibroblasts or epithelial cells spread onto the bottomof 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.
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Focal adhesions are sites where cells adhere to their substratum and send signals to the cell interior
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proteoglycans
Transmembrane proteoglycans also interact with matrix constituents such as other proteoglycans or collagen act as low-affinity growth factor receptors.
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Dr Aliwaini
Epithelial sheets and cell junctions
• 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, toform multicellular sheets
Dr Aliwaini
Functions
• 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
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• 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 othermacromolecules. These include a laminin protein
Dr Aliwaini
Dr Aliwaini
Basement membranes 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).
• 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
Hemidesmosomes
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)
.
• 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
Cell to cell
• Selectins1- E-selectin, present on endothelial cells2- P-selectin, present on platelets and
endothelial cells3- L-selectin, present on leukocytes
(white blood cells).
Selectins depends on Ca2+ ions
We are taking a bout something like what?
Dr Aliwaini
Cadherins
• 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
Cadherins
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)
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Adherens Junctions and Desmosomes:Anchoring Cells to Other Cells
• Adherens junctionsIn an adheren junction, cells are held together by calcium-dependent
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
Desmosomes• 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
TIGHT JUNCTIONS
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 spaceso 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
The main role of junctions
• 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 betweenendothelial cells in a blood vesse.
- 2- Transport : Gap junctions, which allow ions, nutrients, and small signaling molecules such as
Dr Aliwaini
In culture ?
• As epithelial cells differentiate in confluent cultures, theycan form an increasing number of desmosomes and, ifsome morphological organization occurs, can form completejunctional complexes of adherens and tight junctions.
Dr Aliwaini
In culture
• 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, aremore easily dissociated
Dr Aliwaini
Notes!
• 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
What type of matrices you can produce?
• Fibrocytes secrete type I collagen and fibronectininto 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
Matrices
• 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
