1. Physiology Seminar
11/2/2013
PowerPoint® Seminar Slide Presentation prepared by
Dr. Anwar Hasan Siddiqui, Senior Resident, Dep't of Physiology, JNMC
Intercellular Connections
and
Molecular Motors

2. Learning Objectives
 Cell Adhesion Molecules
 Intercellular Connections.
 Brief description of each type and their
function.
 Molecular motors. What are they and what
they do?

3. Cell Adhesion Molecules (CAMs)
 Important cell surface proteins molecules
promoting cell–cell and cell–matrix
interactions.
 Important for many normal biological processes
-embryonic cell migration, immune system
functions, wound healing.
 Involved in intracellular signaling pathways
(primarily for cell death/survival, secretion etc.)

4. Cell Adhesion Molecules (CAMs)
 Express 3 major domains:
• The extracellular domain allows one CAM to bind to
another on an adjacent cell.
• The transmembrane domain links the CAM to the
plasma membrane through hydrophobic forces.
• The cytoplasmic domain is directly connected to the
cytoskeleton by linker proteins.

5. Cell Adhesion Molecules (CAMs)
 Interactions between CAMs can be mediated by
:
Binding of an
adhesion molecule on
one cell to the same
adhesion molecule on
a second cell
Cadherin - cadherin
An adhesion molecule
on one cell type binds
to a different type of
cell adhesion molecule
on a second cell
Selectins – mucins
The linker molecule in
most cases is
Laminin, a family of
large cross shaped
molecules with
multiple receptor
domains.

6. Principal classes of cell-adhesion molecules
 Identified by using specific monoclonal
antibodies (mAbs).
 genes encoding these molecules has shown that
they are structurally different from each other.
 These cell adhesion molecules can be divided
into 4 major families
• The cadherin superfamily
• The selectins
• The immunoglobulin superfamily and
• The integrins

7. The Cadherin superfamily
 Cadherins are the most prevalent CAMs in
vertebrates.
 125 kD transmembrane glycoproteins - mediate
intercellular adhesion in epithelial and
endothelial cells by Ca2+ dependent homophilic
adhesion.
 Primarily link epithelial and muscle cells to
their neighbors
• Form desmosomes and adherens junctions
 Play critical role during development (cell
sorting).
 Do not interact with extracellular matrix.

8. The Cadherin superfamily
 Contain a short transmembrane
domain and a relatively long
extracellular domain containing four
cadherin repeats (EC1-EC4), each of
which contains calcium binding
sequences
 Cadherins interact with specific
cytoplasmic proteins, e.g., catenins
(α, β and γ), as a means of being
linked to the actin cytoskeleton.
 The binding of cadherins to the
catenins is crucial for cadherin
function.

9. The Cadherin superfamily
 E-cadherin is thought to be important during
embryonic development, and is also involved in
generating and maintaining epithelial layers in
adult tissues.
 The loss of E cadherin expression has been
linked to the invasive behavior of tumour cells

10. The Cadherin superfamily
Ca binds in the hinge regions between cadherin
domains, and prevent the flexing. Without Ca the
molecule is floppy and adhesion fails

11. The Selectins
 Involved in heterophilic cell-cell interactions.
 Family of Ca+2 dependent carbohydrate
binding proteins, mediate the initial attachment
of leukocytes to the endothelium on the blood
vessel wall during the rolling step of leukocyte
extravasation in inflammation.
 Selectins recognize fucosylated carbohydrate
ligands, especially structures containing Sialyl-
Lewis x (sLex) and Sialyl-Lewis a (sLea), which
are heavily expressed on neutrophils and
monocytes

12. The Selectins
 Structural features of selectins
include:
• NH2-terminal C-type Ca2+
dependent lectin like binding
domain, which determines the
ability of each selectin to bind to
specific carbohydrate lingands.
• an epidermal growth factor-like
region.
• a number of repeat sequences.
• a membrane-spanning region and
• a short cytoplasmic region

13. The Selectins
 Selectin family
• Leukocyte-expressed L-selectin(CD62L)
• Endothelial-expressed E-selectin(CD62E)
• P-selectin(CD62P) which is expressed by both platelets and
endothelial cells

14. The Selectins
• Recently elevated levels of L-selectin have been observed in the
serum of patients with AIDS and leukemia (1)
• E selectin has been found to regulate adhesion of human colon
cancer cells to the endothelium by binding to sLea and sLex
carbohydrate ligands (2)

15. Immunoglobulin Superfamily Molecules
 Have a series of globular Ig-like
domains, formed by disulfide bonds.
 Mediate Ca-independent cell adhesion.
 Primarily homophilic cell-cell adhesion but also
some heterophilic.
 Activate intracellular signaling pathways.
 Play critical role during morphogenesis and
differentiation of muscle, glial and nerve cells
 In neurons promote the formation of myelin
 In vascular endothelial cells leukocyte adhesion
and extravasation.

16. Immunoglobulin Superfamily Molecules
 Consists of more than 25 molecules.
 Important ones being:
• Intracellular adhesion molecule 1(ICAM1; CD54)
• Intercellular adhesion molecule 2 (ICAM2),
• Vascular cell adhesion molecule1 (VCAM1; CD106),
• Platelet endothelial cell adhesion molecule 1 (PECAM
1; CD31) and
• the mucosal addressin cell adhesion molecule 1
(MAdCAM1).

17. Immunoglobulin Superfamily Molecules
leukocyte endothelial cell
adhesion, endothelial
cell-endothelial cell, and
leukocyte-leukocyte
adhesion

18. The integrins
 Cell adhesion receptors responsible for the cell
extracellular matrix adhesion
 Important signal transduction receptors for
regulation of cell growth
 Present in membranes of all cells except
erythrocytes.
 Composed of heterodimers consisting of two
non-covalently associated subunits,α and β, both
of which are necessary for adhesive binding.

19. The integrins
 Fifteen different α and eight
different β subunits give rise to over
twently different heterodimeric
combinations at cell surfaces.
 Bind epithelial and muscle cells to
laminin in the basal lamina
 Allow platelets to stick to exposed
collagen in a damaged blood vessel
 Allow fibroblasts and white blood
cells to adhere to fibronectin and
collagen as they move

20. Intercellular Connections.
OCCLUDING JUNCTIONS
• Tight Junctions (Zona Occludens)
ANCHORING JUNCTIONS
Actin filament attachment sites
• Cell- cell junctions (Zonula Adherens)
• Cell-matrix junction (Focal Adhesions)
Intermediate filament attachment sites
• Cell-cell junction (Desmosomes)
• Cell-matrix junction (Hemidesmosomes)
CHANNEL FORMING JUNCTIONS
• Gap junctions
SIGNAL RELAYING JUNCTIONS
• Chemical synapse

21. Tight Junctions
 Also known as Zona Occludens.
 Surround the apical margins of the cells in
epithelia such as the intestinal mucosa, the walls of
the renal tubules, and the choroid plexus.
 Made up of ridges—half from one cell and half
from the other—which adhere so strongly at cell
junctions that they almost obliterate the space
between the cells.
 Permit the passage of some ions and solute in
between adjacent cells (paracellular pathway) and
the degree of this ―leakiness‖ varies, depending in
part on the protein makeup of the tight junction.

22. Tight Junctions
 Basic architectural principle - transmembrane
proteins are linked to a cytoplasmic plaque that is
formed by a network of scaffolding and adaptor
proteins, signalling components and actin-
binding cytoskeleton

23. Tight Junctions
 TRANSMEMBRANE TIGHT JUNCTION PROTEINS:
 Tight Junctions contain two principal types of
Transmembrane protein components – tetraspan
and single-span transmembrane proteins.
 The tetraspan proteins are:
• occludin and the claudins
• have both the N- and C-termini in the cytosol.
• form the paracellular permeability barrier and determine
the capacity and the selectivity of the paracellular
diffusion pathway.
 The single-span transmembrane proteins are the
junctional adhesion molecules (JAMs),

25. Functions of Tight Junctions
 Paracellular permeability:
• allow the passive selective diffusion of ions and small
hydrophilic molecules through the paracellular
pathway across epithelia and endothelia.
• the claudin composition of TJs is a major determinant
of the permeability properties of a tissue.
• Occludin regulates the paracellular diffusion of small
hydrophilic molecules, and regulates the
transepithelial migration of neutrophils.
• The passage of solute depends upon its size and
charge.

26. Functions of Tight Junctions
 Cell proliferation, polarity and differentiation:
• Several studies have linked TJs to the regulation of
cell proliferation and cell polarity.
• Occludin suppresses oncogenic Raf-1 signalling
(Wang et al., 2005) and interacts with
ZONAB, thereby regulating gene expression, cell
proliferation and epithelial morphogenesis (Matter
and Balda, 2007; Sourisseau et al.,2006)
• Occludin has also been linked to the regulation of
various subcellular signalling pathways, such as MAP-
kinase-dependent pathways.

27. Disease of Tight Junctions

28. Gap Junction
 Gap junctions are clusters of intercellular
channels that allow direct diffusion of ions and
small molecules between adjacent cells.
 At gap junctions, the intercellular space narrows
from 25 nm to 3 nm.
 gap junctions were first discovered in
myocardium and nerve because of their
properties of electrical transmission between
adjacent cells (Weidmann 1952; Furshpan and
Potter 1957).

29. Gap Junction
 The intercellular channels are formed by head-to-head
docking of hexameric assemblies (connexons) of
tetraspan integral membrane proteins, the connexins
(Cx) (Goodenough et al. 1996).

30. Gap Junction
 Electron microscopy of gap junctions joining adjacent hepatocytes
in the mouse. The gap junction (GJ) is seen as an area of close
plasma membrane apposition

31. Function of Gap Junction
 The diameter of the connexon channel is normally about
2 nm, which permits the passage of ions, sugars, amino
acids, and other solutes with molecular weights up to
about 1000 Dalton.
 Function as suppressors of somatic cell mutations -loss
of a critical metabolic enzyme or ion channel in one cell
compensated by its neighbours.
 Are particularly important in cardiac muscle: the signal
to contract is passed efficiently through gap
junctions, allowing the heart muscle cells to contract in
tandem.
 A gap junction located in neurons referred to as an
electrical synapse are important in neurotransmitter
release

32. Disease associated with Gap Junctions
 20 different genes code for connexins in humans, and
mutations in these genes can lead to diseases that are
highly selective in terms of the tissues involved.
 In humans, mutations in Cx32 underlie X-linked
Charcot-Marie-Tooth syndrome, a common peripheral
demyelination neuropathy.
 mutations in Cx47 result in a central demyelinating
condition.
 disorders of the skin and the auditory system
accompany mutations in Cx31 andCx30.
 Familial cataracts are commonly associated with
mutations in either Cx46 or Cx50.

33. Desmosomes
 Also known as macula adherens is a cell structure
specialized for cell-to-cell adhesion.
 Are molecular complexes of cell adhesion proteins and
linking proteins that attach the cell surface adhesion
proteins to intracellular keratin cytoskeletal filaments.
 The cell adhesion proteins of the desmosome, desmoglein
and desmocollin, are members of the cadherin family.
 On the cytoplasmic side of the plasma membrane, there
are two dense structures called the Outer Dense Plaque
(ODP) and the Inner Dense Plaque (IDP).
• The Outer Dense Plaque is where the cytoplasmic domains of the
cadherins attach to desmoplakin via plakoglobin and plakophillin.
• The Inner Dense Plaque is where desmoplakin attaches to the
intermediate filaments of the cell.

34. Desmosomes

35. Hemidesmosomes
 Hemidesmosomes look like half-desmosomes
that attach cells to the underlying basal lamina.
 Rather than using
desmogleins, hemidesmosomes use
desmopenetrin cell adhesion proteins,which are
members of Integrin family.
 The integrin molecule attach to one of many
multi-adhesive proteins such as
laminin, resident within the extracellular
matrix, thereby forming one of many potential
adhesions between cell and matrix.

36. Molecular Motors
 Molecular motors composed of motor proteins.
 These proteins bind to a polarized cytoskeletal
filament and use the energy derived from
repeated cycles of ATP hydrolysis to move
steadily along it
 Power movements of subcellular components
 Create local forces leading to cell shape changes
• Muscle contraction
 Power cell movements

37. Molecular Motors
 Dozens of different motor proteins coexist in
every eucaryotic cell.
 They differ in the type of filament they bind to
(either actin or microtubules), the direction in
which they move along the filament, and the
―cargo‖ they carry.
 There are three super families of molecular
motors:
• kinesin,
• dynein, and
• myosin.

38. Molecular Motors, Kinesin
 The conventional form of kinesin is a doubleheaded
molecule that tends to move its cargo toward the ―+‖
ends of microtubules.
 Dimer of two heavy chains
 Each heavy chain complexes with a light chain
 Three domains
• Two globular head domains
• Long central coiled-coil stalk
• Two small globular tail domains
(contain light chains

39. Molecular Motors, Kinesin
 Kinesin accomplishes transport by "walking" along a
microtubule. Two mechanisms have been proposed to
account for this movement.
• In the "hand-over-hand" mechanism, the kinesin heads step past
one another, alternating the lead position.
• One head binds to the microtubule and then bends its neck while
the other head swings forward and binds, producing almost
continuous movement
• In the "inchworm" mechanism, one kinesin head always
leads, moving forward a step before the trailing head catches up.

40. Molecular Motors, Dyenin
 Dynein transports various cellular cargo by "walking"
along cytoskeletal microtubules towards the minus-end
of the microtubule.
 Composed of two or three heavy chains (that include the
motor domain) and a large and variable number of
associated light chains.
 Dyneins can be divided into
two groups:
• cytoplasmic dyneins and
• axonemal dyneins, which are
also called ciliary or flagellar dyneins.

41. Molecular Motors, Dyenin
 Cytoplasmic dyneins are found in all eucaryotic cells -
important for vesicle trafficking, and for localization of
the Golgi apparatus near the center of the cell.
 Axonemal dyneins, are highly specialized for the rapid
and efficient sliding movements of microtubules that
drive the beating of cilia and flagella.
 Dyneins are the largest of the known molecular
motors, and they are also among the fastest: axonemal
dyneins can move microtubules in a test tube at the
remarkable rate of 14 μm/sec

42. Molecular Motors, Myosin
 Myosins comprise a family of ATP-dependent motor
proteins and are best known for their role in muscle
contraction and their involvement in a wide range of
other eukaryotic motility processes.
 Most myosin molecules are composed of a
head, neck, and tail domain.
• The head domain binds the filamentous actin, and uses ATP
hydrolysis to generate force and to "walk" along the filament
towards the barbed (+) end (with the exception of myosin
VI, which moves towards the pointed (-) end).
• the neck domain acts as a lever arm for transducing force
generated by the catalytic motor domain.
• The tail domain generally mediates interaction with cargo
molecules and/or other myosin subunits. In some cases, the tail
domain may play a role in regulating motor activity

43. Molecular Motors, Myosin
 18 different families (identified by genetic
analysis)
 Have different functions
• Myosin II powers muscle contraction and cytokinesis
• Myosins I transport of endocytic vesicles
• Myosin V phagocytosis and transport of cellular
elements
• Myosins VI and VII – transport endocytic vesicles
into the cell. Found in the inner ear and mutations in
the gene coding for myosin VII cause deafness in mice
and humans.

44. Molecular Motors, Myosin
 A myosin II molecule is composed of two heavy chains
(each about 2000 amino acids long (green) and four
light chains (blue).
 the long coiled-coil tail bundles itself with the tails of
other myosin molecules forming bipolar ―thick
filaments‖ that have several hundred myosin heads,
oriented in opposite directions at the two ends.

45. Molecular Motors, Myosin
 How does the myosin move?
 Cyclic attachment and detachment of myosin head from
actin filament, each coupled to hydrolysis of one ATP
 ATP binding to myosin opens the cleft and disrupts
actin binding
 Release of actin from myosin head
 ATP hydrolysis - bending of the head to the new
position (generation of movement)
 After ATP hydrolysis the cleft closes on the next actin
molecule

46. ………………Thank You
Any Queries?