CELL BIOLOGY

CYTOSKELET
ON

gy

lo
t
Bio rtmen
pa

De

Drs. H.A.Sofy
Permana,MSc.,DSc.

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BIOLOGY DEPARTMENT
University of Brawijaya
MALANG
Company

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Topics :

Introduction
Structure of Cytoskeletal filaments
Intermediate Filaments (IFs)
Microfilaments (Actin Filament)
Microtubules (MTs)
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Introduction

The cytoskeleton is one of several biological

elements that define eukaryotic cells (others
include the nucleus and mitochondria)

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Cytoskeleton
Means cell skeleton
Internal framework of cell
Has many functions
Anchoring cell organelles
Provide cell shape
Aids in cell motility
Response to environmental signals
Comprises
Microtubules
Microfilaments (Actin filament)
Intermediate filaments
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Inter-cellular
Anchoring

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Cytoskeleton
functions
Provide cell shape ?
How do you think about
The structure of these shapes?

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Cytoskeleton
How do you think about
The structure of these
shapes?

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the shape of cells

pentagonal hepatocyte

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Introduction
Cytoskeleton (from Greeks):
Cyto + skeleton

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Eukaryotic Cells
possess
Cytoskeleton

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How do
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prokaryotic

Cytoskeleton like-filaments in
prokaryotic cells

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Cytoskeleton like-filaments in
prokaryotic cells (CreS)

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Cytoskeleton like-filaments in
prokaryotic cells (MreB)

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Cytoskeleton like-filaments in
prokaryotic cells (FtsZ)

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Cytoskeleton like-filaments in
prokaryotic cells

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Structure and Function

CYTO
SKELETON

Structures:
1. Intermediate
Filaments
2. Actins
3. Microtubules
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Functions:
1. Mitosis
2. Cell Morphol
ogy
3. Cell Motility

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Like our body, the cells

also have skeleton

Actin Filament / Microfilament (Actin)

Intermediate Filaments (IFs)

Microtubules (MTs)

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Eukaryotes Cytoskeleton
Components
1
Intermediate
Filaments
(IFs)

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Microfilaments/
Actin Filaments
(Actin)

Microtubules
(MTs)

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Cytoskel
eton
structur
es

diameter = 8 nm

diameter = 25 nm

diameter = 10 nm
Cell was fixed and stained
by Coomassie blue

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cytomusculature
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Bone Cells

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Osteoblasts
Bone formers. Formation of
bone through ossification or
osteogenesis. Collagen
produced by E.R. and Golgi.
Released by exocytosis.
Precursors of hydroxyapetite
stored in vesicles, then released
by exocytosis.
Ossification: formation of bone
by osteoblasts. Osteoblasts
communicate through gap
junctions. Cells surround
themselves by matrix.
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Osteocytes
Osteocytes. Mature bone
cells. Stellate. Surrounded
by matrix, but can make
small amounts of matrix to
maintain it.
Lacunae: spaces occupied
by osteocyte cell body
Canaliculi: canals occupied
by osteocyte cell processes
Nutrients diffuse through tiny
amount of liquid surrounding
cell and filling lacunae and
canaliculi. Then can transfer
nutrients from one cell to the
next through gap junctions.
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Hyaline Cartilage

Structure: large amount of collagen fibers evenly distributed

in proteoglycan matrix. Smooth surface in articulations
Locations:

Found in areas for strong support and some flexibility: rib

cage, trachea, and bronchi, articular cartilage
In embryo forms most of skeleton
Involved in growth that increases bone length
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Elastic Cartilage
Structure: elastic and
collagen fibers
embedded in
proteoglycans. Rigid
but elastic properties
Locations: external
ears and epiglottis

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Fibrocartilage
Structure: thick collagen fibers distributed in
proteoglycan matrix; slightly compressible and very
tough
Locations: found in areas of body where a great deal of
pressure is applied to joints

Discs of knee joint, pubic symphysis, intervertebral

discs

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Structur
e of
Intermed
iate
Filament
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The IFs are in fully polymerized state,

with very little free tetramers

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Intermediate filaments aspect

an example of how a cell culture is made from tissue.

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Family proteins of
Intermediate
filaments

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Family proteins of
Intermediate
- helical rod domain
filaments
N-terminus

C-terminus
epithelium &
derivates

keratins
vimentin

mesenchymal origin

neurofilament proteins

neurons
eucaryotic cells

nuclear lamins

regions containing heptad repeats

Bundles glial filaments (green)

in cultured astrocytes

Keratin in rat
kangaroo epithelial
cells (Ptk2 cells)

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Family proteins of
Intermediate
filaments
Glial filaments
in glial cell

Neurofilaments
in axon

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Nuclear lamina
in frog oocyte

Actin
filament
structure

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Actin filament structure

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Actin in musclecells
(flash-back)

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Actin in muscle-cells(flashback)

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Actin filament formation

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Actin filament formation

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Actin, myosin and cell

How does a cell move ?
movements

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Cell movements Mechanism

1.Extension
Phase

Text

4.De-adhesion
Phase

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2.Adhesion
Phase

Cell
movements

3.Translocation
Phase

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Actin mesh-work in filapodia

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Actin, myosin and cell

Cell movements model
movements

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functi

Myosin, the actin motor-protein

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Myosin - Actin model

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Myosin - actin experiment

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Myosin, the actin motor-protein

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Microtubule structure

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Microtubulestructure

PROTOFILAMENT

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tubulin
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Schemes of microtubulebstructure

8nm

plusend

minusend

Tubulindimer

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protofilamen
t

25nm
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Tubulin Microtubule structure

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The Microtubule Organizing

Centre (MtOC) in centriols

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Gamma-tubulin ring complex

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Polymerization, the key process of

cytoskeletons self-assembly

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Microtubule formation

animation by ASAHI Miho

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(Nagoya Univ.Jpn)

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Microtubule and motor protein

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MTs - motor protein experiment

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MTs motor protein and Cargo

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MTs - motor protein model

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Dynamic Instability of MTs

is
is Single
Single MT
MT behavior
behavior ((in
in vivo
vivo and
and in
in vitro
vitro))

repeat & randomly change

In length
by undergoing

Growing
phase

Transition
phase:

Shrinking
phase

-catastrophe
-rescue
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Various Regulation factors for microtubule dynamics

Polymerizationfactors

Nucleationenhancer
TuRC

Tubulinactivator
CRMP2etc.

Depolymerizationfactors

Severingfactor

,p56,p48(EF1)et
katanin

Kinesinthatacceleratecatastrophe
Kin13(KIF2A,XKCM1,MCAKetc.

Polymerizationaccelerator
chTOGp/XMAP215etc.

Rescueaccelerator

heatstableMAPsuchasMAP2

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Tubulininactivator(sequesteringfactor)
stathminfamily,HPC1/syntaxin1Aetc.

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Dynamic instability in vivo (in the cell extract)

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Dynamic instability in vivo (in the cell system)

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Dynamic instability in vivo (in the cell system)

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Dynamicinstabilityinvitro(inthefreecellsystem)

by dark-field
microscopy
10m

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DynamicinstabilityProfile

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Howdothecatastropheandrescuein
MTdynamicsoccur?

rescue

catastrophe

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Detaileddynamicinstabilityobservation of single microtubule

minus end

plus end

plus end
minus end

[ItohandHotani,1994]

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Bar=5m

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Microtubule Associated Proteins

(MAPs) is MTs regulator

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Regulation of dynamic instability

Polymerizationofbulkmicrotubules

Suppressionofcatastrophe,
Enhancementofrescueetc.

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Enhancementofcatastrophe,
Severingofmicrotubuleetc.

Depolymerizationofbulkmicrotubules

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Regulation factors for microtubule dynamics 1

CRMP2bindstofreetubulinandincreasesitspolymerizing
activity,butitisreleasedfrommicrotubuleafterpolymerization.

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Regulation factors for microtubule dynamics 2

XMAP215bindstoplusendofmicrotubuleandguidesfree
tubulinstoincreasepolymerizationvelocity.

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Regulation factors for microtubule dynamics 3

Stathminbindstotwofreetubulindimersandsequestersthem
fromthepolymerization.

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Regulation factors for microtubule dynamics 4

HeatstableMAPssuchasMAP2,tauandMAP4bindsto
microtubulelateralsurfaceandincreasesrescuefrequency.

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regulation
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factor

MTsstructureinFlagella
10m
Chlamydomonassp

10m

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AXONEME

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MTsstructureinCilia
Paramaeciumsp

10m

TripletMTsinbasalbody
20m

DoubletMTsincilia

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5m

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on

HowdoCiliaandFlagellamove?
10m

10m

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Cytoskeleton Functions
inmitosis(drosophylaearlyembryo)

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FLUORESCENCE MICROSCOPY
Specimen is excited with a specific wavelength of light, then fluorescent emissions are
observed.

Drawbacks
Photo bleaching can significantly cause measurement error

When to use Fluorescence microscopy

Used to study specimens, which can be made to fluoresce.

Certain material emits energy detectable as visible light when irradiated with
the light of a specific wavelength. The sample can either be fluorescing in its
natural form like chlorophyll and some minerals, or treated with fluorescing
chemicals.
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FLUORESCENCE
Sample you want to study is itself the light source.
Main uses:
Imaging and quantification
Assaying antigens in antigen/antibody reactions
Imaging and quantification of intracellular DNA
Analysis of chromosomal abnormalities

Principle of Fluorescence
1. Energy is absorbed by the atom which becomes excited.
2. The electron jumps to a higher energy level.
3. Soon, the electron drops back to the ground state, emitting
a photon (or a packet of light) - the atom is fluorescing

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Cytoskeleton Functions
inmitosis(frogembryo)

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Cytoskeleton Functions
inmitosis(lungcell)

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DIFFERENTIAL INTERFERENCE CONTRAST MICROSCOPY (D.I.C)

Transforms minute differences in refraction indexes of light passing through an
unstained specimen, or optical path differences from the specimen surface shape, into a
monochromatic shadow-cast image enabling observation.
3D-pseudo effect that it gives and also, unlike phase contrast there are no halos around
the subject
Drawbacks
DIC utilizes optical path differences within the specimen (i.e.: product of refractive index
and geometric path length) to generate contrast the three-dimensional appearance may
not represent reality
Birefringent specimens such as those found in crystals may not be suitable because of
their effect upon polarized light. Similarly, specimen carriers, such as culture vessels,
Petri dishes, etc., made of plastic may not be suitable
When to use DIC?

As with phase contrast microscopy, DIC microscopy may be used with living
specimens. However, it is better suited to thicker specimens.
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The DIC set-up consists of:

A Polarizer,a Wollaston prism the Object, Wave train, Objective, Wollaston prism,
Analyzer, and Eyepiece.

DIC

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Main uses:
Imaging fibrous structure of nerve
Imaging mitotic spindles
Imaging cellular nucleic structures or
other thick unstained specimens

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Cytoskeleton Functions
inmitosis(breastcancercell)

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Cytoskeleton Functions
inmitosis(plantcell)

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Cytoskeleton Functions
Mitosisanimation

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Cytoskeleton Functions
incellmorphology(redbloodcell)

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Cytoskeleton Functions
incellmorphology(euptrellia)

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Cytoskeleton Functions
incellmorphology(amoeba)

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Cytoskeleton Functions
incellmotility(keratocyte)

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Cytoskeleton Functions
incellmotility(spermsmove)

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Cytoskeleton Functions
incellmotility(cilia)

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Cytoskeleton Functions
incellmotility(neutrophil)

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Cytoskeleton Functions
incellmotility(neutrophilexperiment)

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Cytoskeleton Functions
incellmotility(fibroblast)

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Cytoskeleton Functions
incellmotility(heartmusclecell)

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Cytoskeleton Functions
incellmotility(listeriainfection)

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Thank
Thank You
You
very
very much.!!
much.!!
See
See U
U

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