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CSB331H1 Lecture Notes - Adherens Junction, Cell Junction, Tight Junction


Department
Cell and Systems Biology
Course Code
CSB331H1
Professor
Maurice Ringuette

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Lecture 2 Notes January 12, 2011
Notes taken verbatim from Alberts 4th and 5 edition unless otherwise noted.
Apical Junctions: Tight and ad`herens junctions
Introduction
Cell adhesion can be subdivided into two broad categories: cell-cell and cell-matrix
interactions. The cell adhesion molecules (CAMs) that mediate these interactions have a variety of
functions beyond serving as static adhesive elements. For examples, some CAMs act as signal receptors
that elicit changes in cell shape and gene expression. Intracellularly, these transmembrane receptors
molecules are linked to complexes that play a direct role in signal transduction pathways. As with signal
transduction pathway, cell adhesion involves a combination of different CAMs acting at the same time.
The information must be integrated for proper development and tissue homeostasis to be maintained In
other words, multiple parallel interactions are occurring at the same time and the information generated
must be integrated.
There here are six major types of tissues:
Nerve
Muscle
Epithelia
Connective Most text classify blood and lymphoid tissues as
Blood connective tissues
Lymphoid
What is the difference between junctional and non-junctional adhesion?
Junctions are often visualized by conventional or freeze-fracture electron microscopy.
In vertebrates, intercellular adhesion of epithelial cells at the apical end is primarily mediated by
two types of apical junctional complexes (APJs): tight junctions (TJs) and adherens Junctions (AJs). At
the basal end, desmosomes serve to anchor epithelial cells to basal laminae [a specialized extracellular
matrix (ECM) sheet we will discuss extensively in later lectures].
Occluding junctions: tight junctions (TJs)
TJs are also often referred to as apical junctions because of their location at the apical end of
epithelial cells. The main function of TJs is to act as paracellular sealants/barriers between adjacent
epithelial cell and endothelial cells, ensuring homeostasis. In multicellular organisms, homeostasis is
dependent of being able to isolate the internal environment from the external environment. In addition,
distinct internal fluid compartments must also be isolated from each other, e.g. lymphatic and vascular
systems. Cellular sheets of epithelia, endothelia and mesothelia establish tissue compartmentalization.
Cellular sheets can be simple monolayers (e.g. gut epithelia) or stratified (e.g. skin), the latter being less
polarized than the former.
Focusing of polarized epithelial sheets, TJs form a branching network of sealing strands that completely
encircles the apical end of adjacent cells in an epithelial sheet (Figures 19-25 and 19-26A). Each TJ
strand is embedded in the lipid bilayer and forms a tight lateral association with another TJ strand on the
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opposing membrane of an adjacent cell, at the apical-lateral end. The resultant paired strands reduce the
intercellular distance to almost zero. The ability of TJs to restrict “paracellular” diffusion increases
logarithmically with the number of strands in the network- suggesting the strand act independently. There
is a positive correlation between the number of strands and the tightness of the epithelium. “Tight” TJs
have on average 5 or more strands, whereas “leaky” TJs often have only one strand. Diffusion between
epithelial cells is referred to as “paracellular” diffusion/secretion.
While TJs function as intracellular sealants to prevent paracellular diffusion of ions
across epithelia, they show ionic charge and size selectivity depending on the cell type and physiological
requirements. At present, two functionally distinct pathways have been identified: a high capacity,
charge-selective pore pathway that permits the passage of small ions and uncharged molecules. The
second is a low- capacity leak pathway that allows flux of larger ions and molecule independent of
charge. Small ions do not discriminate between the pore and leak pathways. As noted in your text, TJs
associated with the gut epithelia are 10,000 times more permeable to Na+ ions than epithelia lining the
bladder wall. Another critical function of TJs is to prevent lateral diffusion (intermixing) of membranes
associated proteins and lipids from the apical to basal end of epithelial cells and vice versa. Moreover TJs
act as regulators of epithelial cell proliferation and differentiation by recruiting to the apical domain
signaling molecules. Indeed, a recurring theme in molecular biology is that most proteins have diverse
context-dependent functions. As our technologies become more sophisticated, their diverse functions
become more evident.
TJs are composed of three classes of evolutionarily conserved transmembrane proteins: claudins,
occludins, and ZO (Zona Occludin) proteins. The best studied is the claudin family, which consists of at
least 24 members in mice and humans and is the focus of course. Claudins are approximately 23 kDa
transmembrane proteins bearing four transmembrane domains. See Fusukita and Furuse (2002) Curr
Opinion Cell Biol 14:531-536 for a more detail description of the structural organization of claudins. Do
not memorize all that information, only the specific molecular components that will be focused on in
class.
Two key points:
1) More than one claudin species is often expressed by a single cell.
2) Heterogeneous claudin species are integrated into a single strand.
In class, I will present experimental evidence by Sonoda et al (1999, J Cell Biol 147: 195-
204) that claudins are involved in TJ strand formation and barrier function. Figure 1shows
immunohistochemical evidence that MDCK (Madin Darby- canine kidney cells) co-expressed claudin-1 and
4. MDCK cells are popular for epithelial studies since they form well polarized epithelial cell monolayers
with intact TJs and are readily transfected with recombinant vectors. This enables researchers to investigate
the effects of over- and under-expressing normal and mutated proteins involved in regulating the
morphogenesis, differentiation and function of epithelial sheets.
Confluent cultures of MDCK cells were stained with anti-claudin-1, claudin-2, claudin-3 or
claudin-4 antibodies. As you can in slide 15, intense claudin-1 and claudin-4 immunostaining is visible at
apical cell borders. Weak immunostaining is visible for claudin-2 and claudin-3, likely due to background,
non-specific staining that is often observed at cell borders.
Clostridium perfringens enterotoxin (CPE) is a 35 kDa polypeptide that binds specifically to
claudins 3 and 4. When MDCK cells are incubated with CPE, claudin-4 begins to disappear from TJs,
resulting in the degradation of the TJs visible after 4 hours (Figure 5, slide 16). Using transwell permeability
assays, they demonstrated ionic charge selectivity and barrier function are also lost. Overexpression of
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