Cell Systems- cells signalling PDF

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Summary

Experimental TechniquesModule learning outcomes:  Demonstrate a critical awareness of the structure-function relationship of cells and tissues  Describe core signal transduction pathways including steroid hormones, G-proteins and tyrosine kinases  Explain how neurons communicate to mediate comple...

Description

Experimental Techniques Module learning outcomes:  Demonstrate a critical awareness of the structure-function relationship of cells and tissues  Describe core signal transduction pathways including steroid hormones, G-proteins and tyrosine kinases  Explain how neurons communicate to mediate complex biological functions  Compare and contrast the anatomical and physiological features of skeletal, cardiac and smooth muscle  Discuss how the different cell types of the immune system mediate immunity  Manipulate and interpret cellular and physiological data Hierarchy

Atoms -> molecules -> macromolecules -> internal cell structures-> Cells-> tissues -> organs -> organ systems -> organism Histology One of the easiest ways to assess cells The study of microscopic structures within tissues- 5 stages  Fixation: fixing cells or tissue into place, could be done with heat- evaporate water off a sample to get it to adhere onto a slide or through perfusion (passage of fluid through blood vessels)- making use of an organisms circulatory system to perfuse a fixative solution through blood vessels or through. The fixative is injected into the heart with the injection volume matching cardiac output. The fixative spreads through the entire body, and the tissue doesn't die until it is fixed. immersion- where a sample can be submerged into formaldehyde to fix and also preserve the specimen- overall process going on for all techniques: water being taken out of system- water allows sample to rot and can be used by proteolytic enzymes to break down sample (which we do not want)  Embedding- trying to retain morphology- e.g. with paraffin wax- suspend sample in wax and cooled, alternative such as: agar, epoxy resins, acrylic can be used to make sample solid- size of samples can be very variable e.g. brain or biopsy sample. The way the 

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sample is embedded can make a difference in what happens next- e.g. whether you want a vertical or coronal slice of brain impacts how you embed a sample Sectioning- can use a microtome: a machine with a very sharp scalpel blade to slice a thin section- microtomes can cut slices that are 5-15um thick, once pieces come off the microtome they are usually suspended in fluid to remove excess paraffin e.g. water bath vibratome- can be used on smaller samples to cut even thinner samples, ultramicrotome- use diamond blade that cut sections that are 50-150 nm thick Staining- most biological material does not have a lot of colour, silver stain can be applied cerebellum to show three different layers that make up cerebellum Visualise- using a microscope to see tissue structures

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The tissues are very static and inherently quite 2D If there are issues in any stages, there will be issues with the final product e.g. distorted cells, distorted morphology, may not have many good sections, staining may not be done well, may struggle to see details in sample on slide

Micro chatter: happens when sample is too brittle, either because too much alcohol during staining process or staining is too cold- either block it is in is too cold or it has been taken from the freezer too recently. Can also happen when the block and blade of microtome aren't secured properly- can see it has been cut badly

Through the middle there is a paler section- much thinner than rest of specimen- potentially because the block moved as it has been cut or machine moved, or there was an issue with fixation so blade moved through it differently

This sample has been damaged by heat or exposure to acid- top image shows bubbling as if it has been cooked, bottom image shows cracking- within cracks, cells are distorted and shrunken hinting that sample has been mistreated e.g. paraffin wax too hot or water bath (used after sectioning) was too hot

There is insufficient staining, not enough stain means you can't visualise much- may not have removed fixative or paraffin wax well enough

Sample has been physically traumatised- it has been damaged before histology occurred – forceps may have gripped edges too hard – often occurs at early part of histology when sample is fresh or has been partially fixed and is still soft- picking this up roughly can cause trauma Light microscopy  Passing light through a sample, or reflecting it off the sample (dissection microscope)  Glass lenses magnify the image

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Variations: Brightfield, darkfield and phase contrast- change the way you can visualise the sample Limited resolution- light scatters away from the focal plane- max 2000 fold magnificationdifficult to distinguish between objects Using 100x objective needs oil- no oil means very low resolution can cannot distinguish between cells as not enough detail

Fluorescence microscopy

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Higher resolution Higher cost More specific training Use fluorescence to highlight structures either within cells or cells in tissues- shining light onto electrons on samples which can jump up to a higher energy level (excited state) when they fall back down to ground state they release a photon which can be detected as fluorescence Need to use specific wavelength of excitation light- use a laser which needs to be kept cool and enough space Still using lenses and mirrors to shine light onto slide and returning through objective Diagram shows- below the photo multiplier detector, there is a pinhole aperture which should be aligned with the focal plane of sample you look at meaning that any light that is not within focal plane is filtered out- increasing resolution With fluorescence, immunohistochemistry can be performed

Immunohistochemistry Immunohistochemistry is based on immunodetection which is accomplished by fluorescence microscopy Silver stained cerebellum

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Three layers 5 types of neurons

Stained with fluorescent antibodies

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Class 3β tubulin- green Glial fibrillary protein- red Cell nuclei –blue

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Can see three layers very clearly Get a better idea of the cells in relation to each other

Can be used to highlight specific proteins within cell  

Antibodies staining for Golgi body (left) Antibodies staining for Endoplasmic reticulum (right)

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Protein of interest (left) overlay with ER (middle) Overlay with (right)

Overlaying of protein with pervious images shows it is not hugely associated with ER, but when overlayed with golgi body, they basically light up the same area => protein of interest has something to do with golgi body and less with ER  Better idea of location=> better idea of function Electron microscopy  Vastly higher resolution (10,000,000 X vs ~ 2,000 X)  Using beam of electrons rather than light that comes from electron gun  Not using lenses to filter wavelengths of light- lenses focus the electron beam using electromagnetism or electrostatics  Because resolution is so high, can get a good view of stellate structure of collagen or microvilli on epithelial layer  Instead of using stains, the contrast can be developed using heavy metals  Electron gun operates as a vacuum, any specimens would get damaged or dehydrated if left in microscope for too long Electrophysiology  Most microscopy is on very static and usually dead cells  Electrophysiology lets us look at activity of live cells  Three main categories- extracellular, intracellular, patch-clamp  All techniques use glass electrodes which have chloride wires which are connected to an apparatus which can detect changes in potential or voltage used to measure electrical activity in cell.  Extracellular: Can follow processes such as release of neurotransmitters  Intracellular: pierce cell to get in cytoplasm- changes in potential across membrane, usually cell cannot be tested over and over  Patch-clap: place electrode close to membrane and apply suction which draws up some membrane into tip of electrode- can record changes in current going through a single ion channel Patch-clamp 

Cell attached, whole cell, inside-out, outside in recordings

SDS-PAGE SDS-PAGE (sodium dodecyl sulphate–polyacrylamide gel electrophoresis), is a discontinuous electrophoretic system which is commonly used as a method to separate proteins  There is a complex, tertiary and secondary protein structure  If you are trying to compare multiple proteins because there are different secondary and tertiary structures, they need to be linearised in to their primary structure  Usually done through heat  To do SDS-PAGE they need to stay linearised which makes use of SDS (sodium dodecyl sulphate) molecule

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SDS has a carbon and hydrogen tail and a negatively charged sulphur group on its head

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way it works- once you have a linear protein, the hydrogen and carbon tails align with it and -ve heads are poking out All protein will stay linearised because SDS blocks it from retwisting back into secondary structures Protein will have net –ve charge Amount of –ve charge corresponds to how long that chain of amino acids is Short aa chain will have a smaller net –ve charge

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This can then be loaded on gel Load sample into well, turn power pack on and proteins will move towards positive electrode

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SDS-PAGE works in a similar way Instead of it moving horizontally along table, vertical power packs are used to drag from top of gel to bottom gel

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Example stained with instant blue Can see ladder on left to compare stains to Instant blue only stains proteins but stains ALL proteins- is not specific to particular protein

If we want to visualise specific proteins, a western blot can be done  Take gel and transfer things on gel onto a membrane (often a nitrocellulose membrane)  Apparatus sandwiches everything together and holds it in place  Once it is on the membrane, specific staining can be done for the specific protein  Antibodies can be used as they are very specific- means that antibodies can be used to detect specific amount of protein of interest in sample- relative proportions

Example: figure above shows western blot gels and below each one, there is one for beta-actin  Beta-actin is constitutively expressed: quantities are the same across every lane which means you can look at amount of EPO changing in comparison to beta-actin  We know that the baseline amount of protein expression is equal because bands of betaactin are all the same density  => changes in density of EPO bands mean something experimentally  Antibodies can be fluorescent  Dont always have to be fluorescent-sometimes secondary antibodies may have an enzyme on it that produce a coloured product so coloured bands are produced  Black bands produced in EPO image is production of a pigment

Example of western blot

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Ligand has been added (epidermal growth factor- EGF) to cells to look at if they will bind to receptor (EGFR) and what effect this will have on RK protein kinase (ERK) signalling pathway Different time points at top with control showing when no EGF has been added at all Amount of phosphorylated EGFR increases as time progresses

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Once receptor is phosphorylated, It means it is activated- once EGF is present, EGFR is activated Then, looking at how this goes on to affect ERK protein kinase In order to check that the results on the phosphorylated ERK western blot are not just due to change in total kinase or accidentally loaded more sample there are two loading controls ERK total kinase loading control GAPDH loading control- functioning like beta-actin- density of bands should all be the same, showing you have not artificially raised the amount of phosphorylated ERK by adding more sample to those lanes- each lane contains same amount of protein Total kinase shows that concentration of ERK isn't changing but the fact that it is being activated or phosphorylated is due to presence of EGF Receptor is phosphorylated for full 16 mins but phosphorylated protein kinase is only active for first 8 mins post exposure and then amount of phosphorylated ERK starts to reduce by 16 mins Time dependent response- protein kinase is not just switched on, once EGF is introduced to system, it is a timed response in response to that ligand Understand the principle of prepping a sample for histology Understand the principles and limitations of light microscopy Describe the advantages and disadvantages of electron microscopy Briefly describe the different type of electrophysiological recordings Understand the process of SDS-PAGE and western blots, and how these can be utilised for cell signalling studies

Epithelium and Connective Tissue Four basic tissue types: 1. Epithelium 2. Connective tissue 3. Muscle 4. Neural Epithelium  

Proper epithelium covers and lines outer and inner body Glandular epithelium forms glands and secretes hormones and other substances

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line our vessels, make up the lining of organs and skin Structure closely related to function- can tell what it does based on how it looks

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Simple epithelium found in places where substances need to diffuse e.g. alveoli are lined with simple epithelium but CO2 and oxygen need to diffuse from alveoli into bloodstream and vice versa which would be difficult with a thick layer of cells

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Stratified epithelium found in places needing to resist chemical or mechanical stress e.g. oesophagus as when food comes through it may be sharp or hot so a stratified epithelium acts as a protective layer of the underlying tissue of the oesophagus

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Epithelial cells are attached to the basement membrane

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The basement membrane is not made of cells but is made up of different types of fibres e.g. collagen The basement membrane is semi-permeable to certain substances which is important because epithelial tissue is avascular (have no blood vessels) and so get nutrients from underlying tissues Nutrients will diffuse from underlying tissue through basement membrane to epithelial cells All epithelial cells are polar Apical side exposed to outside body or the internal cavity that is it lining Basal side (inner side) is tightly attached to the basement membrane (a thin layer of mostly collagen fibres that helps hold epithelium together and anchor it to connective tissue) Many boundaries are selectively permeable allowing for some level of absorption, filtration and secretion of substances

Single squamous (squamous= flat)  For fast absorption and diffusion, making thin membranes  Cells take a lot of time and energy and raw materials to make so in places where a lot of cells are lost such as outer skin or mouth, there are more squamous cells since they are cheaper than big cuboidal or columnar ones  

Squamous cells in a single layer Built on a basal lamina

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Single layer= permeable (effective for Gas, diffusion, filtration since they are so thin) and delicate Only found in protected locations within the body, not things that would be exposed to trauma or stress Lines capillaries, alveoli, glomeruli as they are very smooth, so don’t get build-up of plaques Shown below- around bigger vessels (main excretory duct, artery) there is a much thicker layer of epithelial cells Around veins and inside of capillaries, a much thinner layer of epithelial cells- (only around one cell thick for capillaries)

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Stratified squamous (stratified= arranged in layers)       

Squamous cells in layers Built on a basal membrane Because they are layered, they are more resistant to damage and trauma- can damage outer layer cells, inner layer cells can then replace them Found in places where mechanical stress is more likely Non-keratinised (left images) (cant dry out)= mouth, eyes, internal membranes Keratin fibres add a protective layer to the cells on the surface- added when need waterproofing and increase resistance to trauma Keratinised (right images) (dry and impermeable) = skin, especially palms and soles, gums, top of mouth (hard palate) and tongue

Simple Cuboidal  Absorb nutrients and produce secretion       

Nucleus no longer squished Single layer on a basal lamina Basal lamina is wrapped around to make a vessel and lined with a single layer of simple cuboidal cells Cells are physically larger than squama cells, in 3D they extend out a lot more meaning they have space to have larger nuclei Still delicate, so found in places that don’t undergo much trauma Common secretive tissues (passive or active release of materials) Found in Kidneys, thyroid gland, eyes, salivary glands, ovaries, testes- places in body that are quite well protected by the rest of the body

Simple columnar  Absorb nutrients and produces secretions

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Even larger and taller Nucleus is found on the basal edge (edge on same side as basal membrane) Still only a single cell thick but because the cell is larger, there is more space for compression and resistance Generally, they are secretory cells, because they are tall there is a lot of space within their structure to have machinery for secretion Often ciliated, cilia are always on apical surface (surface facing out) Cilia are able to move things passed cells (e.g. mucus, liquid) and increase surface area If there is absorption going on (e.g. in stomach) there is a larger surface area for interacting with nutrients Simple columnar line digestive tract (stomach, small and large intestine)

Stratified columnar

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Found in places with high levels of mechanical stress but also where secretion takes place Stratified columnar found in salivary glands, conjunctiva (eyes), pharynx (back of throat), anus and male urethra

Columnar cells make up stomach lining as they have more space than squamous cells therefore have more internal machinery to make and secrete mucus Pseudostratified and ciliated   

Pseudostratified- looks like it is stratified because the nuclei are in different locations but in fact it is still only a single layer of cells made up from more than one cell type Ciliated columnar cells moves mucus and other liquids in respiratory tract, airways, fallopian tubes, uterus and central spinal cord Pseudostratified= contain > 1 cell type so looks like multiple layers, but a single layer

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Pink epithelial cells, blue goblet cells, connective tissues

You can tell from the size and shape of the epithelial cells, what they are doing and about their function  Flat cells generally involved in absorption or gas exchange  Taller cells usually involved in secretion  Stratified cells generally in places that experience stress Glandular epithelium  Epithelium tissue that secretes a substance= glandular  Can be Single cells or even complex organs  Size of the cell or community of cells isn't a good indicator of how important it is e.g. pituitary gland is very small but controls huge amount of what goes on in developing and adult body, damage to this has a big impact on organism  Can be arranged into complex structures- endocrine and exocrine glands  Endocrine= secrete substance into extracellular space, circulated in the blood e.g. pancreas, thyroid, pituitary gland  Exocrine= secrete into a duct, taken directly to another organ or the surface of an epithelium e.g. sweat glands, salivary glands, lacrimal glands (in eyes that produce tears), sebaceous glands (on skin that produce sebum)  (duct= tube or pipe)           

Generally when cells are organised into a gland, they follow similar patterns Such as the ones sh...