The Cytoskeleton, Cilia & Undulipodia + Homogenization, fractionation and cytology Flashcards
What is the cytoskeleton?
- Fibrous network - to which organelles are tethered
- Provides structure and organisation
- Cytosol approx 55% total cell volume
- 20% of the cytosol is proteins
NOT FOUND IN BACTERIA OR ARCHAEA - EUKARYS ONLY
What is the cytoskeleton structure?
- Cytoskeleton proteins including:
- Microfilaments (actin)
- Myosin (types 1 and 2)
- Microtubules (tubulin)
- Intermediate filaments (cytokeratins)
- Intermediate filaments anchor organelle in cells
What does cytoskeleton play a major role in?
Cytoskeleton plays a major role in maintaining cell structure and shape
Role of the cytoskeleton
- Cell shape and mechanical resistance to deformation
- Stabilisation of tissues, through association with extracellular connective tissue and other cells
- Cell migration (contraction of cytoskeleton)
- Endocytosis & intracellular transport (movement of vesicles and organelles within the cell)
- Chromosome segregation during mitosis and meiosis
- Cytokinesis
- Contraction of muscles
- Form specialised structures, such as eukaryotic flagella, cilia, lamellipodia and podosomes
What are the microfilaments in cytoskeleton?
- 2 strands of intertwined actin - 7nm in diameter
- Cell shaper - tension bearing load
- Muscle contraction
- Cytoplasmic streaming
- Cell motility
What are the Actin in cytoskeleton?
- G actin is a 5nm diameter globular protein that can polymerise to form F actin which has a diameter of 7 nm
- F actin has 2 intertwined polymer chains of G - actin that form a right handed double helix with 13 actin monomers per turn
- F actin microfilaments have +ve ends where polymerisation occurs and -ve ends where actin is lost
(Polymerisation requires hydrolysis of ATP)
( Polymerisation it is controlled by capping proteins)
What does myosin 1 do?
- Myosin 1 is a ubiquitous cellular protein - functions in vesicle transport
- Myosin 1 has a globular head with ATPase activity and a short tail which can bind to other proteins
- Myosisn 1 can attach to organelles including the endoplasmic reticulum, Golgi and vesicles and ‘walk along’ F - actin microfilaments carrying organelles
What is myosin 2 responsible for?
Myosin II (also known as conventional myosin) is the myosin type responsible for producing muscle contraction in muscle cells in most animal cell types
What promotes muscle contraction?
Nerve impulses cause calcium release which promotes muscle contraction
What are intermediate filaments?
- Size approx 10nm
- Structural/mechanical strength of cells and tissues
- Cable - like structure
- Compromised of a variety of proteins (no single polymers)
- Found only in vertebrates
- more stable (strongly bound) than microfilaments
Intermediate filament proteins?
-There are about 70 different human genes coding for various intermediate filament proteins
-However they share the same characteristics – they are all polymers that measure between 9-11 nm in diameter when fully assembled
-Animal IFs are subcategorized into six types based on similarities in amino acid sequence and protein structure
Type 1 and 2 intermediate filament proteins
These proteins are the most diverse among IFs and constitute type I (acidic) and type II (basic) IF proteins.
The many isoforms are divided in two groups:
-Epithelial keratins (about 20) in epithelial cells
-Trichocytic keratins (about 13) (hair keratins), which make up hair, nails, horns and reptilian scales
What are type V intermediate filament proteins?
- Provide structural function and transcriptional regulation in the cell nucleus
What is the assembly of intermediate filaments?
- Single polypeptide chains - wind to form tetramers
- Organised in anti - parallel fashion
- Filament is comprised of 8 protofilaments
- IFs more stable - lack dynamic movement
- Phosphorylation - regulates function (nuclear envelope during mitosis) - Lamins
What are the cell junctions in animal cells?
Provide the link between neighbouring cells, tissues and
organ systems
Cell junctions and their features in animal cells?
1) Tight Junctions
* plasma membranes are tightly bound by proteins that form a continuous seal around cells that is water tight
2) Desmosomes
* act like protein rivets that anchor cells together
* desmosomes link to sturdy cytokeratin* fibres that project into the cytoplasm
3) Gap junctions
* provide cytoplasmic channels between cells allowing cell functions to be linked in synchrony such as heart muscle and smooth muscle contractions
What is cytokeratin?
Cytokeratin is a keratin rich intermediate filament
Type 1 and II IFS
What is the intracellular organisation of IFs (Type V)
- Complex network in the cytoplasm
- Extends from plasma membrane to the nucleus
- Keratin/ vimetin anchor the nucleus within a cell
Integrates all aspects of the cytoskeleton (actin/ microtubules)
Cytoskeleton in the context of disease?
- Duchenne muscular dystrophy (DMD)
- X - linked disorder
- Mutation in the gene encoding dystrophin
Dystrophin links the cytoskeleton with certain transmembrane proteins in muscle cells. It plays an important role in stabilising muscle cells so that they don’t slide too much and cause strain related muscle damage.
Micro tubule structure
- Alpha tubulin (purple)
- Beta tubulin (blue)
- 13 pro - filaments, comprised of tubulin dimers, arrange to form cylindrical microtubule
- Dynamic process of growth and shrinkage
- Mediated by GTP hydrolysis
Microtubule structure (2)
- Microtubules can have 3 structures:
O singlets in cytoplasm and mitotic spindles
OO doublets in cilia and flagella
OOO triplets in centrioles and basal bodies
Microtubule - role in the cell cycle
- Mitosis - partition of replicated chromosomes
- Involves the assembly and disassembly of a key microtubule structure - mitotic apparatus or mitotic spindle
Cell cycle During prophase
- During prophase, assembly of spindle microtubules begins in the centrosome, the microtubule organising centre
- The centrosome replicates, forming two centrosomes that migrate to opposite ends of the cell, as spindle microtubules grow out from them
What are centrioles?
- Centrioles have 27 stable microtubules organised into 9 + 0 triplets surrounded by a protein matrix
- Centrioles pairs are organising centres that form microtubule spindles during mitosis
What are the centrioles and the centrosome?
- In many cells microtubules grow out from a centrosome near the nucleus
- The centrosome is a microtubule organizing centre
- In animal cells the centrosome has a pair of centrioles each with 9 triplets of microtubules arranged in a ring
DO NOT CONFUSE CENTROMERE WITH CENTROSOME THE CENTROME IS A REGION OF THE CHROMOSOME
WHILE THE CENTROSOME IS A MICROTUBULE ORGANIZING CENTRE
The mitotic spindle
- In anaphase, sister chromatids separate and move along the kinetochore microtubules toward opposite ends of the cell
- The microtubules shorten by depolymerising at their kinetochore ends
- Microtubules are dismantled by the kinetochore to release tubulin subunits
Microtubules – organelle movement
Axonal transport of vesicles along microtubules in nerve axons involves kinesin and cytosolic dynein motor proteins:
vesicle can be transported 250 – 400 mm/day
Kinesin /dynein head proteins have ATPase activity and tails that bind to vesicles:
kinesin mediates anterograde movement of vesicles towards synapses along singlet microtubules
cytosolic dynein mediates retrograde movement to the cell body for recycling along singlet microtubules
- In synaptic endings motor proteins can transport neurotransmitter vesicles along actin microfilaments
What does kynesin do?
Kynesin
- Anterograde transport (towards + end of microtubule
- Transport of cellular cargo
- Important role in depolymerising microtubule minus ends at centrosomes during anaphase
What does Dynein do?
Dynein
- Retrograde transport (towards - end of microtubule
- Transport of cellular cargo
- Cytoplasmic dynein: organelle function, helps to position the Golgi complex and other organelles in the cell, centrosome assembly
- Axonemal dynein: cilia/flagella movement
What do cilia and flagella do?
- Flagellum undulate in a snake like motion driving the cell forward e.g. human sperm
- Cilia move back and forth with a rapid power stroke and slower recover stroke
What do cilia and Flagella share ( common ultrastructure)
- Cilia and flagella share a common ultrastructure:
- A core of microtubules sheathed by the plasma membrane
- A basal body that anchors the cilium or flagellum
- A motor protein called dynein which drives the bending movements of a cilium or flagellum
How cilia/ undulipodia beat?
Dyneins are arranged in doublets on the inside flagellum/cilium
Dynein ‘walk’ along microtubules, carrying cargo (microtubule)
Dyneins release from microtubule, swing forward, and re-bind
ATP required
Inhibitory signals on either side of flagella - switch inhibition mechanism
As one side is inhibited, other side is active - beating motion
Dyneins release from microtubule, swing forward, and re-bind
What do proteins cross links limit?
Sliding
Forces exerted by dynein arms cause doublets to curve, bending the cilium or flagellum
What must you do in order to obtain information about cells/organelles?
To obtain precise information about the cells
organelles, it is necessary to isolate them free
from contamination organelles
Cell Homogenisation and fractionation involves 3 steps?
It involves 3 steps: Extraction, Homogenisation and fractionation
What happens at the extraction phase?
It is the first step toward isolating any sub-cellular structures.
In order to maintain the biological activity of organelles and biomolecules.
The cells or tissues are suspended in a solution of appropriate pH and
salt content, usually isotonic sucrose (0.25 mol/L) at 4°C.
What is homogenisation?
- Homogenisation just means to “break open”.
- Homogenisation is concerned only with rupture of
the membrane.
Principal aim of homogenisation?
The principal aim is to achieve:
* highest degree of cell breakage
* minimum of disruptive forces
* without damage to any of the organelles
Homogenisation Cell Disruption Methods
The choice of method?
- Volume/number of cells to be homogenised?
- Number of samples to be homogenised?
- How difficult are the cells to homogenised?
- What impact will the method have on the desired
product? - How stable is the product being isolated?
- How dangerous are the cells, products and
methods used? - Cost of method?
The choice of method should take these
factors into consideration and to
ultimately achieve the best product yield
and quality.
What is a bead mill?
- Bead mill homogenisers are mechanical solid shear homogenisers that agitate beads at high speeds to break up cells
- Agitators are fitted with the shaft which
provide kinetic energy to the small beads
present in the chamber - Disruption takes place due to the grinding
action of the rolling beads and the impact
resulting from the cascading ones. - The choice of bead size and weight is
greatly dependent on the type of cells
What is Ultra Sonication
- Ultrasonic homogenisers mechanical liquid shear
work by inducing vibration in a titanium probe
combined with an aspirator device that is immersed
in the cell suspension. - Homogenisation occurs both directly through the
ultrasonic forces and cavitation - the rapid formation
and collapse of bubbles producing a shockwave and
disrupting cell walls by pressure change. - This combination of forces make ultrasonic
homogenisers excellent for cell disruption.
Homogenisation
Cell Disruption Methods
1 - 8
Ultra Sonication benefits + drawbacks?
- Perfect when sample is mostly liquid or consists of
small particles in a liquid. - Unsuitable for applications where sample is mostly
solid and / or not immersed in a liquid buffer.
What is French Press
- mechanical liquid shear which consists of a
hydraulic pump that drives a piston. The piston
forces the liquid sample through a tiny valve under
high pressure. - This pressure could be between 10,000-50,000psi
- As the sample passes through the
valve, the cells experience shear
stress, resulting in cellular
disruption. - As the cells move through the valve,
they experience decompression and
subsequently expand and rupture
What is Thermolysis
- Non-mechanical physical which mainly used for
bacteria. - Involves heating the cells (heat shock) to 50-55C to
disrupt the outer membrane and release periplasmic
proteins (Gram –ve) - or up to 95C to break the cell wall and release
cytoplasmic contents (E coli, Gram +ve). - Relatively easy and cheap but used only for heat
stable products
What is osmotic shock?
- Osmotic shock is a non-mechanical physical, it is
osmotic stress which is physiologic dysfunction
caused by a sudden change in the solute
concentration around a cell, - This causes a rapid change in the movement of
water across its cell membrane (in/out) result in cell
burst. - Note that this method can only work with animal
cells and protozoa, since they do not have cell walls
(bacteria).
What is Chemical Solvent
- It is a non-mechanical (chemical) mainly used in plant cells.
Organic solvents such as toluene, ether, benzene, methanol,
and DMSO (Dimethyl sulphoxide) can be used to permeate
cell walls. - EDTA can be used specifically to disrupt the cell walls of gram
negative bacteria, whose cell walls contain
lipopolysaccharides. EDTA will chelate the cations leaving
holes in the cell walls.
What is Detergents
- It is non-mechanical (chemical) , which directly
causes damage to the cell wall or membrane. - Its mechanism of action is to solubilise membrane
proteins and lipids, thereby causing the cell to lyse - Detergents can be anionic, cationic and non-ionic
detergents. - Most commonly used anionic detergent is sodium
dodecyl sulfate (SDS) which disturbs protein-protein
interactions.
What is Enzymes method
- It is non-mechanical enzymatic which degrade the
cell wall which will lead to release the intracellular
components. - Enzymes that are commonly used for degradation
of cell wall of plants (cellulases), yeast
(zymolyases), Grame –ve/+ve (lysozomes), and
animal cells (collagenase and proteinase K). - The enzyme’s high price and limited availability
limits their utilization in large scale processes
What is Subcellular fractionation
It is the process used to separate cellular
components/organelles while preserving
individual functions of each component
How is Subcellular fractionation carried out
Mainly carried our using a centrifuge which is a
spinning device which applies high-speed centrifugal
forces to separate materials based on density/size.
What is Principle of centrifugation
- Particles suspended in a solution are pulled
downward by Earth’s gravitational force. - In a solution, particles whose mass or density is
higher than that of the solvent sink or sediment and
particles that are lighter than they float on the top
Centrifugal Methods
fractionation/separation
of organelles
- Separation by – Differential centrifugation
- Separation by– Density gradient centrifugation
What is differential centrifugation
- Differential centrifugation: separates particles based
on difference in sedimentation rate, which reflect
differences in sizes and densities of organelles
Steps of Differential centrifugation
1-The preparation (homogenate) is initially
centrifuged at low speeds to completely sediment the
largest and heaviest sub-cellular component.
2- The supernatant is carefully decanted and is again
centrifuged at a higher speeds till the desired portion
of cell lysate is obtained
What is Density gradient centrifugation
Density gradient centrifugation: separates
particles in which the sample is centrifuged in
a medium that gradually increases in density
from top to bottom.
What is commonly used for
dentistry gradient
Sucrose (5-40%) is commonly used for
dentistry gradient
What happens in density gradient configuration?
- The homogenate is applied in a thin zone at the
top of the centrifuge tube on a density gradient
(sucrose). - If the density of the organelle at any point in
the gradient is same to that of the gradient,
then these organelles will stop otherwise it will
move downward towards more denser region
What are Marker Enzymes
- An enzyme that is known to be localised exclusively in the
particular organelle. - Marker enzymes also provide information on the
biochemical purity of the fractionated organelles. The
presence of unwanted marker enzyme activity in the
preparation indicates the level of contamination by other
organelles.
Allows for one to monitor the fractionation
of organelle protocol
What is cytology
Is the microscopic examination of cells
obtained from the body by aspiration or
scraping for diagnostic purposes,
Main advantage of cytology
The main advantage of this test is that the
collection of cytological specimens does not require
anaesthesia, is painless and there is no bleeding
neither inflammation.
Components of the microscope
1-Stage: flat platform where slides are placed
2- Ocular Lens (10x): where specimen viewed though
3-Objective lens: Lens closest to the specimen; changes
magnification
4-Base: support microscope and contain light source
5-Diaphragm (Iris): Adjust the amount of light that
reaches specimen.
6-Coarse Focus: will bring specimen into a general focus.
7-Fine Focus: will fine tune the focus, increasing the
detail.
8-Light Source: white light source found on base.
9-Condenser: focus light onto specimen.
10-Mechanical stage arms: move stage L, R, up and down
Fluorescence Microscopy
- Major Function: Localisation of specific
cellular molecules – example proteins
Fluorescent dyes or proteins
(Flurochromes)
* flurochromes may be indirectly or directly
associated with the cellular molecule
* Multiple flurochromes may be used
simultaneously
- Major Advantages: Fluorescence Microscopy
- Sensitivity: “glow” against dark
background - Specificity: immunofluorescence
- Cells may be fixed or living
Processing cytology samples steps
- Sample collections
- Reception of the specimen and request form.
- Preparation of slides for microscopic.
- Staining
- Screening and reporting the slides.
Cytology Sampling Techniques
1-Exfoliative cytology
2-Fine needle Aspiration Cytology (FNAC)
3-Body fluids
1-Exfoliative cytology
Microscopic examination of cells that have been
shed or removed from the epithelial surface
(mucous membrane) of various organs.
2-Fine Needle Aspiration Cytology (FNAC)
is a diagnostic procedure used to investigate lumps or masses
(a way of taking a sample of cells from the breast tissue).
A thin (23–25 gauge), hollow
needle is inserted into the mass
for sampling of cells
- Body fluids:
is a diagnostic procedure in which a thin (23–25 gauge),
needle is inserted into the organ cavity for sampling of
cells.
Pleural and pericardia fluid, CSF
and ascitic fluid
Cytology specimens
- peritoneal, pericardial and pleural fluids
- CSF
- Nipple discharge
- Bronchial brushings / washings
- Sputum
- Gastric washings
- Urine sediment
- Prostatic secretions
- Cervicovaginal (paps) smear
- Reception of the specimen and request form.
*Details on the specimen and form are checked.
*The specimen and form must have
at least three patient identifiers
(out-of-name, surname, date of birth, NHS number, hospital
number).
*Laboratory reference number assigned.
- Preparation of slides for microscopic 2 techniques
- Line smear technique
- Squash technique
Preparation of slides for microscopic
* Line smear technique
- Place a drop of fluid near the end of a slide and drag a spreader slide backward
into the drop. - The drop spreads along the junction of the two slides.
- When the spreader slide reaches no more than 2/3 the length of the slide, lift it
directly upward.
- Preparation of slides for microscopic.
- Squash Technique
Place a drop of fluid near the frosted end of the slide.
Gently place a second (spreader at 90 degree) slide on top of the drop
slowly pull the spreader slide across the first slide
- Staining:
- prevention of degeneration of cells and tissue
- preservation of cells as close as possible to the living state
- specific periods of time
- changes the physical and chemical state of the cells
Appropriate fixative agent:
- Penetrate cells rapidly
- Minimize cell shrinkage
- Maintain morphologic integrity
- Deactivate autolytic enzymes
- Replace cellular water
- Facilitate diffusion of dyes across cell boundaries
- Help cells adhere to a glass surface
- Provide consistent results over time
Common fixatives:
- 95% Ethyl alcohol
- 100% Ethanol
- 100% methanol
- 80% isopropanol
What is Permeabilisation
involves treating specimens with a mild
detergents (such as saponin and digitonin,
Tween 20) to dissolve lipids from the cell
membrane, thereby allowing stains to dye
intracellular components and organelles
What are the 2 Principle of Staining
- Basic (+ve) = stain the acidic components of
the cell (nucleus) - Acidic dye (-ve) stains the basic components of the
cell (cytoplasm)
Steps of Staining
*Fixation
*Permeabilisation
*Hydration: to prepare the cell sample for uptake the nuclear dye
*Staining
*Clearing: cells are not transparent while the smear is in the staining or alcohol solutions. During clearing, Xylene replaces the alcohol , which is also miscible in the mounting medium.
*Mounting: slides are cover slipped with a mountant that bonds them and
protects the sample from air drying, oxidation, or fading of the stain.
3 ways of staining?
Nuclear staining: is done by using a hematoxylin stain.
Dehydration to prepare the cells for cytoplasmic staining
Cytoplasmic staining: cytoplasmic stains are Eosin, OG-6/EA-50.
What is Papanicolaou (Pap) staining
It was developed by Dr Papanicolaou by 1940s
It is a polychrome stain which to this day is used
It is one of the most used staining procedures in cytology
What is Hematoxylin & Eosin
(H & E) staining
For routine diagnosis, the use of H & E is by far
preferred for viewing cellular and tissue structure
detail by pathologists.
What is Eosin
Eosin is an acidic
dye: it is negatively
charged. It stains
basic (or acidophilic)
structures red or
pink.
What is Hematoxylins
Hematoxylins is a basic
dye, it is positively
charged used to stain
acidic (or basophilic
structures nucleus)
blue.
What is Diff-Quik staining
It is known as Romanowsky stain.
is based on a modification of the Wright-Giemsa stain
Is used for cytoplasmic element detail, elements such as mucins,
fat droplets and neurosecretory granules.
Diff-Quik stain is often used for initial screening of cytopathology
specimens.
Diff-Quik allows the pathologist to quickly decide whether or not
additional staining is required
What is Immunocytochemistry
- Detection of surface antigens (markers) on isolated cells.
- The detection is based on specific antigen-antibody binding
(immunoreactions). - A specific antibody identifies an epitope with 8-15 length amino
acid sequence in a protein (epitopes).
What is immunocytochemistry used for?
- Tumour diagnostic/classification
- Prognostic/Predictor Markers
- Target Therapy
Cytology in practice
Cervical screening
