Mcp Lect 21: The Cytoskeleton And Cell Junctions

Front 1

Structure (components) and function of the cytoskeleton?

Back 1

Function:
-internal scaffolding of the cell
-responsible for the shape of cells
-driving force behind cell movement

Structure: Consists of three major components:
1. intermediate filaments
2. Microtubules
3. Microfilaments
(main difference between the 3 is size!)

Front 2

Diameter of microfilaments?

Back 2

5 nm

Front 3

Diameter of microtubules?

Back 3

25 nm

Front 4

Diameter of intermediate filaments?

Back 4

7-11 nm

Front 5

Intermediate filaments - general characteristics

Back 5

-ropelike polymers of coiled-coil fibrous polypeptides
-relatively stable
-cell type specific --> different types of intermediate filament proteins are associated with different cell types
**this characteristic can be used for typing cells (eg. keratins are markers for tumors of epithelial origin)

Front 6

keratin

Back 6

a. intermediate filaments
b. markers for tumors of epithelial origin

Front 7

vimentin

Back 7

a. intermediate filament
b. marker for tumors of connective tissue cells (sarcomas)

Front 8

Lamins

Back 8

intermediate filaments!

**lamins a,b, c !!

Front 9

Intermediate Filament Groups

Back 9

4 major groups based on the STRUCTURE of the polypeptide subunits:
Type I - IV

Front 10

Intermediate filament assembly

Back 10

The central domains of two individual proteins (a-helices -- repeat for 7 amino acids -- heptad repeats) form a helical dimer

Two dimers line up side by side to form a tetramer

Tetramers aggregate end to tend to form a protofilament

Eight protofilaments form a cylindrical, 10 nm thick intermediate filament

The globular domains at either end project from the surface and accounts for variations between individual types of intermediate filaments

Front 11

Intermediate filament function?
where in the cell are they found?

Back 11

-Help to maintain cell structure
-extend from the nuclear envelope to the plasma membrane
-they are stable, ie no evidence for a rapid polymerization or depolymerization
-particularly prominent in cells that are subject to mechanical stress, such as epithelial and cardiac muscle cells
-keratin filaments form a network that crisscross the interior of the cell and attach to SPOT DESMOSOMES

Front 12

Keratin filaments are major structural components of?

Back 12

Keratinized structures such as the outer layer of the skin, hair, and nails
-- keratin filaments form a network that crisscrosses the interior of the cell and attaches to spot desmosomes

Front 13

General characteristics, composition of microtubules?

Back 13

-single tubules are 25 nm in diameter
-composed of 2 monomers - a- and b-tubulin, which polymerize to form a hollow single microtubule 25 nm in diameter
-assembly is regulated by microtubule-associated proteins (MAPs)
-Microtubules have a defined polarity
-Assembly and disassembly occurs preferentially on one end
-Occurs as single, double, or triple tubes

Front 14

Assembly of the (protein subunits) of microtubules is regulated by?

Back 14

Assembly of the two monomers -- a- and b- tubulin -- which make up microtubules is regulated by MICROTUBULE-ASSOCIATED PROTEINS (MAPs)

Front 15

Which of the microtubule ends grows at a slow rate? At a fast rate? What is this regulated by?

Back 15

– end is where microtubule grows at slow rate, + end is end where dimers added more rapidly

Regulated by microtubule-associated proteins (MAPs)

Front 16

What is this an image of?

Back 16

Microtubule single, double, or triple tubes

In the doublet, tube B shares part of the complete tube A

In the triplet, tube B shares parts of both the complete tube A and the complete tube C

Front 17

Microtubules originate from?

Back 17

microtubule organizing center, which contains centrioles that are perpendicular to each other

-microtubules radiate away from this region forming a star like structure termed an aster
-amorphous material in this region serves as a nucleating site (or primer) for the formation of cytoplasmic microtubule

Front 18

Functions of microtubules ?

Back 18

3 of the major functions of microtubules:
1. make up single apparatus during mitosis where chromosomes line up at equator of cell and are pulled apart as cell divides. Part of spindle apparatus
2. used to transport materials from one part of cell to periphery.
3. Make up structure of flagella and cilia, projections of cell involved in cell motility

Front 19

Kinesin

Back 19

-composed of heavy and light chains
-used to walk cargo on microtubules TOWARD THE + END

have globular heads that bind to microtubules, and hydrolyze ATP and tails that bind to transported vesicles

Front 20

Dyenin

Back 20

-composed of heavy and light chains
-used to walk cargo on microtubules TOWARD THE - END

have globular heads that bind to microtubules, and hydrolyze ATP and tails that bind to transported vesicles

Front 21

What proteins walk cargo on microtubules?

Back 21

kinesin (--> + end)
dyenin (--> - end)

*both proteins have globular heads that bind to microtubules, and hydrolyze ATP and tails that bind to transported vesicles

Front 22

Distribution and function of microtubules?

Back 22

-present in almost all vertebrate cells
-form a diverse array of both permanent and transient structure

1. cytoplasmic microtubules are transient in nature
-principle structural elements of the cytoplasm during interphase and the mitotic spindle during mitosis
-region surrounding the centrioles is termed the MICROTUBULE ORGANIZING CENTER (MTOC)
-provide tracks for movement of pigment granules and membrane vesicles
*kinesin and dyenin
-responsible for axonal transport in neurons
-during mitosis bundles of microtubules radiate from the poles of the cells and attach to the chromosomes to form the mitotic spindle -- these microtubules are responsible for separating chromosomes during mitosis

Front 23

What microtubules make up the mitotic spindle?

Back 23

-astral
-kinetochore
-polar
microtubules

Front 24

Centrioles

Back 24

-a centriole located at the base of a ciliary axoneme is termed a BASAL BODY
-centrioles can convert into basal bodies and vice verse
-both centrioles and basal bodies consist of 9 sets of triple microtubules (1 coplete 2 incomplete) forming a short cylnder 0.2 um x 0.3 um
-centrioles usually arise by the duplication of preexisting ones

Front 25

basal body

Back 25

-base of cilia
-serves as a nucleation site for the growth of axoneme microtubules
-centrioles (formed from microtubules) can convert into basal bodies and vice verse

Front 26

Structure / size of basal bodies v. centrioles?

Back 26

-both centrioles and basal bodies consist of 9 sets of triple microtubules (1 coplete 2 incomplete) forming a short cylnder 0.2 um x 0.3 um

Front 27

Clila function

how do they move?

Back 27

extend from the surface of many epithelial cells, which line the trachea, oviduct, and parts of the male reproductive tract and function to move fluids, mucous, eggs, and sperm

similar structure to flagella

move via DYENIN WALKING

Front 28

Flagella function

Back 28

function to propel sperm

similar structure to cilia

move via DYENIN WALKING

Front 29

dynein walking

Back 29

Dynein is a protein associated with individual microtubules in an axoneme

the dynein arms on one set of microtubule doublets interact with adjacent microtubule dublet and hydrolyzes ATP to generate a sligind force between the two sets of microtubules

sliding of microtubule doblets is converted into bending of the cilia or flagella -- microtubule doubles are held together by a network of cross-links

Front 30

Axoneme

Back 30

AXONEME:
2 singlets in the center
+
9 doublets surrounding RADIAL SPOKES
+Nexin (connects each doublet together)
+Dynein arms

make up cilia and flagella

Front 31

Microfilament general characteristics

Back 31

-very dynamic
-rapid if polymerization and depolymerization
-RESPONSIBLE FOR CELL MOVEMENT
-present in all vertebrate cells
-can account for a significant fraction of total cellular protein

Front 32

Microfilament structure

Back 32

-composed of a helical chain of ACTING

Front 33

F- actin

Back 33

filamentous actin

polarized and grows faster on one end than the other

Front 34

G-actin

Back 34

monomeric actin (globular)

Front 35

Proteins that interact with actin to modify its behavior:

Back 35

1. cross linkers
-- some proteins can cross-link actin filaments into 3d networks
--this is responsible for the reversible gel-sol transitions of actin
--may be an important factor in cell movement

2. Fragmenting (or capping) proteins which prevent polymerization, regulating the loss or addition of G-actin -- regulated by Ca2+ ions

*these proteins are part of the actin filament and can change the structure and function of the actin filament

Front 36

Gel-sol transitions of actin?

Back 36

gel --> cytosol is gel like
sol --> cytosol flows freely

Front 37

Myosin

Back 37

-represent a family of proteins that interact with actin (microfilament) to produce tension
-can bind to actin and posess an ATPase
-ATP hydrolysis is essential for movement
-two major forms have been described:
1. Type 1 (aka minimyosins)
2. Type 2 myosin

Front 38

Type 1 myosin
v
Type 2 myosin

Back 38

TYPE 1:
aka minimyosins
-present only in NONMUSCLE CELLS
-contains a short tail that can bind to membrane vesicles, plasma membrane, and microfilaments
-responsible for movement of organelles along actin filaments

TYPE 2:
-present in BOTH MUSCLE and NON MUSCLE CELLS
-contains a gobular head that can bind actin and a fibrous tail that can form polymers
-in the case of striated muscle the myosin filaments are organized into thick filaments and bind to F- actin

Front 39

Functions of Actin:

Back 39

1. intracellular movement
2. muscle contraction
3. membrane structure (microvilli in intestine and sterocilia in choclea of ear and epidimius)
4. locomotion (cortical flow / leading edge)

Front 40

Where does actin interact with the plasma membrane?

Back 40

1. Zonula adherens or intermediate junctions
2. Cell cortex

Front 41

Molecules that mediate cell adhesion:

Back 41

There are several major families of cell adhesion proteins -- these include:
1. Integrins
2. Immunoglobulin superfamily
3. Selectins
4. Cadherins

Front 42

Integrins

Back 42

-family of heterodimers
-function both cell-substratum and cell-cell adhesion
-include the fibronectin receptor
-attached to the cytoskeleton
-Ca2+ dependent
-cell survival signal -- lose connection via integrans, lose signal

particlar a and b subunit determin what ECM protein an integrin binds to (fibronectin, laminin, collagen ,etc binding depends on subunit)

Front 43

Immunoglobulin superfamily

Back 43

-cell-to-cell adhesion during embryogenesis, wound heling, and inflammatory response
-includes N-CAM, the first adhesion molecule to be described

have Ig like domains

-homophilic binding!

Front 44

N-CAM

Back 44

-medites the fasciculation of axons
- important in the attachment of neurons to skeletal muscle cells
-immunoglobulin superfamily
-homophilic binding

Front 45

Selectins

Back 45

-bind specific carbohydrates on target cell
-expressed by some white blood cells and by endothelial cells
-mediates an inflammatory response in which leukocytes bind to blood vessle

-allows cells to move with respect to one another!

Front 46

Cadherins

Back 46

-Ca2+ dependent
-homophilic cell-cell adhesion molecules
-developmentally regulated
-mediated cell-cell adhesions by serving as anchoring sites for actin filaments (intermediate junctions)
-present on most cell types and bindts to itself
-Dimers from one cell + dimers from another cell
-can be divided into a number of subtypes depending on their distribution and cell binding specificity
**junctions can form between cells having the same subtype of cadherin but not between cells having different subtypes

**may mediate cell sorting
**may control morphoigenesis

CATENINS AND OTHER PROTEINS MEDIATE CONNECTION OF CADHERIN TO ACTIN

Front 47

You can us Ab to block what kind of cell-to-cell adhesion?
what's another way you can achieve this same effect?

Back 47

Ab to cadherins prevents intermediate junctions
eg. e-cadherins

can also us CALCIUM 2+

Front 48

What holds most epithelial sheets together?

Back 48

e-cadherin

-abundant at sides of cell-to-cell contact in epithelia
-addition of antibodies to this cadherin cause cultured epithelial cells to detach from each other

Front 49

What molecule may mediate cell sorting?

Back 49

cadherins
-when different embryonic tissues are dissociated into individual cells and then allowed to reaggregate, they sort out, ie cells from different tissues segregate into different regions of the aggregate
(Can happen between tisseuss with different cadherins, ie. n-cadherin and e-cadhering, or between cells that have high/low levels of the same cadherin)

Front 50

What molecule may control morphogenesis?

Back 50

cadherins!

during development, the expression of the various cadherins undergoes dynamic changes

the loss of cadhering may be responsible for the epithelial to mesenchymal transformation

different germ layers express different forms of cadherins which may account for their separation

Front 51

Zonula adherens aka

Back 51

Intermediate Junctions

Front 52

Intermediate junctions aka

Back 52

Zonula adherens

Front 53

Cell cortex

Back 53

a layer of actin and associated proteins which is immediately beneath the plasma membrane of many cells
-attached to the plasma membrane by linker proteins
-gives mechanical strength to the cell surface and enables the cell to change shape and to move
-the thickness varies from cell to cell and between different regions of the same cell

Front 54

Function of intermediate junctions

Back 54

1. Structural -- maintain cell surface structures
a. microvilli of the intestine
b. stereocilia of epididymis (aids absorption) and in cochlea (hearing)
2. Muscle contraction
3. Movement -- cortical flow and leading edge model

Front 55

Cortical Flow Model

Back 55

-one of two models (other being leading edge model) that describes how actin filaments help cells move

-controlled by the flow of actin filaments in the cell cortexs
-this flow is governed by the gradients of TENSION in the cortex generated by myosin (flow low --> high tension)
-myosin may be responsible for maintaining the tension
-during initial stages of cell division, there is an increase in the tension over the entire cell surface
-as cell division progresses, the tension at the two polar regions is relaxed, however, tension remains high in the equator leading to an indentation
-tension is controlled by the MITOTIC SPINDLE
-the contractile ring is a bundle of actin filaments and associated myosin located immediately below the plasma membrane of the cleavage furrow
-contains a higher concentration of actin than other areas of the cell surface
-cell surface components flow into the cleavage furrow during cytokinesis

**bundling of actin filaments in one portion of the cell leads to relaxation of the other portion of the cell

Front 56

Leading edge model

Back 56

*mechanism accounting for cell migrationhave cell and get protrusion of leading edge in direction that cell wants to move. Little feet = focal contacts, constantly being formed and broken. In the leading edge model have polymerization of actin occurring in the lamellipodum.. + end.. Extends lamellipodoium then get flow of actin in that direction then focal contact in back of cell breaks. In that way get translocation/locomotion of cell due to polymerization of actin in leading edge of cell and formation of new focal contacts in front and breaking of old in back.

Front 57

Communicating gap junctions:
distribution
morphology
function

Back 57

Distribution:
-on epithelial cells and a variety of other cells, especially those that are electrically coupled (smooth and cardiac muscle)
-most COMMON type of junction

Morphology and composition:
-under EM, membranes of adjacent cells are separated by a 2 nm gal (and channel is 1.5 nm in diameter)
-Connexons have diameter such that it allows ions, small molecules through, but there is NO ACTIVE TRANSPORT PROCESS GOING ON HERE.5000 and 20000 MW protein cannot pass through connexons but 100 and 1000 MW protein can
-composed of several hundered connexons
-each connexon is composed of six subunits of a dumbbell-shaped protein called connexin

Function:
-allows small molecules to pass from cell to cell
-the pore size is large enough to pass ions, sugars, and amino acids, but small enough to block the passage of proteins, nucleic acid, and complex carbohydrates
_may be important for transmitting nutrients (metabolic cooperation)
-can transmit a wave of depolarization from cell to cell
-electrical coupling is important for contraction of heart and smooth muscle cells
-regulates the coordinated beating of cilia on epithelial sheets

Front 58

Proteins making up gap junctions?

Back 58

-composed of several hundered connexons
-each connexon is composed of six subunits of a dumbbell-shaped protein called connexin
2nm space (?)
12 connexins total

Front 59

Zona occludens aka

Back 59

Tight (impermeable) junctions

Front 60

Tight (impermeable) junctions

Back 60

Zona occludens

Front 61

Proteins making up zona occludens ?

Back 61

occludin and claudin

Front 62

Zona occludens

Distribution
Morphology
Function

Back 62

aka tight (impermeable) junctions

Distribution:
found between epithelial cells

Morphology:
-membranes between adjacent cells appear to fuse
-freeze fracture shows a series of parallel strands of intramembrane particles
-specific proteins in adjacent cells make contact with each other to form the tight junction
**flow of tracer is blocked by tight junctions
-made of 2 proteins: claudin and occludin
-appears to involve Ca2+ dependent proteins

Function:
serves as PERMEABILITY BARRIER so that material cannot pass between cells
-functionally divides the plasma membrane of cells into apical and basolateral regions
-many membrane proteins cannot pass this junction
-requires divalent cations

Front 63

Desmosomes aka

Back 63

Macula Adherens

Front 64

Macula Adherens aka

Back 64

Hemidesmosomes

Front 65

Desmosomes & Hemidesmosomes

Distribution
Morphology
Function

Back 65

aka Macula Adherens

Distribution:
-found between adjacent epithelial cells
-button like points of contact between cells
-transmembrane linker proteins connect the two cytoplasmic plaques
-hemidesmosomes in contact with basement membrane

-morphology: contain a pair of dense attachment plaques separated by a 20 nm space
-otfen has a dense line in the center
-KERATIN FILAMENTS fun into the plaque and then course back
-there are filaments between the plaques of adjacent cells

Front 66

Hemidesmosomes are located where???

Back 66

in regions of epithelial cells in contact with the basement membrane and anchor the cells to the extracellular matrix of the basement membrane

Front 67

Gap between desmosomes?

Back 67

20 nm space between the plaques

Front 68

Protein components of the desmosome?

Back 68

cytoplasmic plaque:
-plakoglobin
-desmoplakin

connecting plaques **20 nm space:
-desmoglein and dexmocollin

Attached to plaques:
KERATIN FILAMENTS

Front 69

Zonula adherens aka

Back 69

Intermediate Junctions

Front 70

Intermediate Junctions aka

Back 70

Zonula adherens

Front 71

Intermediate junctions

Distribution
Morphology
Composition

Back 71

aka Zonula adherens

distribution:
-occurs as a continuous band around the apical portion of epithelial cells
-similar structures occur in intercalated disks of heart muscle

Morphology;
-contain two plaques into which actin filaments insert
-EM shows an ill defined material in the zonula adherens connecting adjacent cells together

composition:
-contains CADHERINS
-family of membrane proteins involved in Ca2+ dependent cell-cell adhesions
-present on the cell surface and are closely associated with the zonula adherens

Front 72

Which of the cell junctions are Ca2+ dependent?

What about the CAMs?

Back 72

-Zona occludens/tight junctions
-Desmosomes
-Zona adherens/intermediate junctions

NOT (to my knowledge) gap junctions

CAMs:
-integrins
-cadherins
-

Front 73

Proteins associated with intermediate junctions?

Back 73

cadherin dimers inside
actin filaments associate with cadherin dimers

Front 74

What mediates the connection of cadherin to actin?

Back 74

CATENINS AND OTHER PROTEINS MEDIATE CONNECTION OF CADHERIN TO ACTIN