Scoil an Leighis agus

Eolaíocht An Leighis UCD

UCD School of Medicine

& Medical Science

Cell Biology, Signalling, and Culture

BASIC CELL STRUCTURE

What does a cell do?

Feed; cells need sugars proteins and minerals, transported over themembrane into the cytoplasm by active and passive means.Excrete; reverse process to feeding!Grow and replicate; complex and tightly regulated process (will discuss further)Migrate; the function of many cell types (such as vascular endothelial cells)depend in part on their ability to migrate/Produce; many cell types produce specific molecules at different times andplaces, ie mature osteoblast secretes calcium/collagen=bone.Degrade; some cell types such as osteoclasts degrade molecules or structuresMake decisions; Grow or die, differentiate or proliferate etc?Communicate; with surrounding cells and tissuesRespond; to autocrine, paracrine and endocrine factors.

All the above processes are controlled by extra- and intra cellular signalling`

Cell Growth

The cell cycle, or cell-division cycle(CDC), is the series of events in a cellbetween one cell division and thenext.

A specialized form of cell division isresponsible for cellular differentiationduring embryogenesis and, as well asfor the maintenance of stem cellsduring adult life.

The cell cycle consists of four distinct phases: G1 phase, S phase, G2 phase(collectively known as interphase) and M phase.

M phase is itself composed of two tightly coupled processes: mitosis, inwhich the cell's chromosomes are divided between the two daughter cells,and cytokinesis, in which the cell's cytoplasm physically divides.

Cells that have temporarily or reversibly stopped dividing are said to haveentered a state of quiesence called G0 Phase, while cells that havepermanently stopped dividing due to age or accumulated DNA damage aresaid to be senescent.

Some cell types in mature organisms, such as Parachymal cells of the liverand kidney, enter the G0 phase semi-permanently and can only be induced tobegin dividing again under very specific circumstances; other types, such asepithelial , continue to divide throughout an organism's life.

Cell Cycle

Apoptosis- Programmed Cell Death

Apoptosis is a process of deliberate liferelinquishment by a cell in a multilcellularoraganism. It is one of the main types ofprogrammed cell death (PCD), andinvolves an orchestrated series ofbiochemical events leading to acharacteristic cell morphology and death.The apoptotic process is executed in sucha way as to safely dispose of cell corpsesand fragments.

In contrast to necrosis, which is a form oftraumatic cell death that results fromacute cellular injury, apoptosis is carriedout in an orderly process that generallyconfers advantages during an organism'slife cycle.

Cell signalling

stimulus

receptor

Signalling pathway response

Signalling involves the transmission of a stimulus (chemical, biochemical orphysical) into a biochemical signal that leads to an intercellular response

Introduction to cell culture

Cell culture is a process in which cells are extracted from a particular tissue,identified and expanded in vitro.

Advantages of cell culture are that cells can be maintained and tested in acontrolled and manipulatable environment, allowing us to study specificfunctions. Also cell culture is generally less expensive and complex than invivo or clinical studies.

The major disadvantage of cell culture is that it is impossible to replicate theprocesses occurring in a complex tissue, as in general we can only work withone cell type at a time.

History of cell culture

-Basic experiments were conducted at the end of the 19th centurywhere tissue explants were maintained in buffer solutions for severaldays-1907-1910 Ross Granville Harrison, a leading embryologist,published results of experiments carried out at Yale; these providedthe basis for cell culture methodologies-1940s-50s cell culture techniques became widespread in virologicalresearch, especially in the production of vaccines. The Salk Poliovaccine became the first vaccine mass produced using Cell Culturetechniques.-One of the first ‘cell lines’ used for cancer research (HeLa) wasdeveloped in 1951, still in use today.

-The work of early cell biologists was hampered by a number offactors;

-Materials- eg. glassware vs plasticware, lack of proper asepticalfacilities-Knowledge- relatively poor knowledge of cell genetics, cellbiochemistry-Techniques- limited methodologies to study cell behaviour

- However in the past 20-30 years there as been an exponentialgrowth in our understanding of cell biology, and consequently cellbiology is now an important area of modern scientific and medicalresearch.

What do we need?

Sterility; bacteria and fungi grow much quicker than mammalian cells, andtherefore avoiding contamination is a constant concern. Cells are culturedin sterile containers, in media containing antibiotics, and all cell work iscarried out in sterile laminar airflow hoods.Plasticware; cells are cultured in sterile, disposable flasks, and all liquidstransferred with sterile pippettes.Media; cells need food! There are a vast array of media available, manytailored to the requirements of specific cell types. Most contain minerals,sugars, sources of proteins and buffering agents. Most medias aresupplemented with animal serum.Incubators; cells are generally grown in a humidified 370C atmosphere with5% CO2.Microscope; inverted or phase contrast microscope.

SterilitySterile, or more accurately aseptic technique is of utmost importance intissue culture

All plasticware used is prepacked, presterilised and disposable.

All cell work is carried out in laminar airflow hoods. These hoods providean area with filtered air so cells can be handled without riskingcontamination.

All areas swabbed/sprayed with 75% isopropanol prior to use.

All waste liquids sterilised immediately (bleach tablet in waste bottle)

Any non-cell culture specific liquids filter sterilised before treating cells,and all non-disposable items (ie glass bottles) autoclaved before use

ContaminationAn annoying, but largely avoidable fact of life!!!

Bacterial Contamination; occurs generally due to contact contamination,ie. Touching pipette of your hand; small cells, difficult to see undermicroscope. Can be countered by supplementing media with antibiotics.

Fungal Contamination; can occur both by contact and spore infection(most common in late summer/autumn); larger cells or visible hyphae.Antibiotics are no use.

If you get contamination review your lab technique and clean all hoods andincubators.

Plates and roller bottles; 98, 24 and 6 well plates are usedfor treating cells; roller bottles are used for large scaleculture of cells

Flasks; 25, 75 and 175cm2 are the standard sizes.Used for culturing and treating cells

Pipettes; 2, 5, 10, 25ml graduated pipettes, used withmechanical pipette aid

Plasticware

Plasticware should be pre-sterile and individually wrapped

Major suppliers include corning and sarstedt

MediaBasal media the defined fractionTypical basal media formulation;

-ammino acids-vitamins-sugars-minerals-buffering agents

Media is supplemented with animal serum (bovine, porcine etc). Serumcontains proteins, growth factors etc.-the undefined fraction-may varyfrom batch to batch.

Many medias contain phenol red; a pH indicator, which turns yellow atacidic pH.

Incubators

Cells must be grown in a humidified atmosphere at 37CSpecialised incubators are available for this;Water and air jacketedSome have 5% C02 for buffering purposesSome of the more modern ones are self cleaning.

MicroscopesCell culture microscopes are ‘inverted’ or ‘phase contrast’ microscopes.There is a different arrangement of lens/light source, with the lens beingbelow rather than above the subject. This allows us to better visualise thecells.

These microscopes are generally up to twice as expensive as regularmicroscopes

Sourcing and Storing Cells

Ultimately all cells are derived from tissue; so called primary culture; often acomplicated and delicate procedure.

Cells can also be bought from cell banks - ATCC - EACC

Cells are stored in freezing media which contains DMSO, this has to beremoved before culturing.

Generally when a new cell line is bought in to a lab as many stocks aspossible are made of it. These are stored in liquid N2. Cells in liquid N2 will‘keep’ for an indefinite length of time.

Growing and Treating Cells

Cells are cultured in their specific media in a flask until they reach desireddegree of ‘confluence’ (expressed as % of surface covered)

Cells are ‘passaged’ or ‘plated’ into fresh flasks/plates. The enzyme trypsinis used to breakdown extracellular matrix and release cells intosuspension.

The passage or ‘p’ number refers to the number of times a particular batchof cells have been passaged; most cell types change with increasingpassage number.

Cells are generally plated into 96/24/6-well plates for treatment. This allowsa number of different experiments to be carried out in the one plate-savesspace cells and media.

Cell stock in 10%DMSO

Thaw and centrifuge Place in flask with fresh media

P2 P2

P3

Incubate at 37C/ 5%CO2.

feed if necessary

Wash cells andincubate with 1xtrypsin EDTA

Spin cellsPlate into new flasks/plate

Treat cellsTime/dose response

Harvest experiment!!!

General lab etiquette applies in the tissue culture lab; you need to takeresponsibility for your own work, and share the responsibility for thesmooth running of the lab.

-make sure your experiment is planned and you have everything youneed and know what your about to do.-clean up after yourself-if the bins/aspirator flasks need emptying, or if stuff needs to beautoclaved, do it your self-if supplies are running low report this-if something is broken report it-don’t use other people’s supplies without permission-if your unsure of something, ASK.

What cell culture can tell us

RNA

Protein

Whole cell

supernatant

Cell function

Gene expression levels

Protein levels,enzyme activity

Protein localization,immunostaining

Levels of secretedprotein

Motility, growth

stimulus

receptor

Signalling pathway response

Studying cell biology in vitro

As mentioned previously we can use cell biology to study the effects ofcertain stimuli and responses; which may have direct clinical relevance

For example, expression of a certain gene may be corellated with increasedagresivity and poorer prognosis in cancer (ie NET1 in gastric cancer)

Or a particular treatment may have serious side effects; ie a number ofclasses of antiretroviral drugs used to treat HIV infection are associated withbone and liver toxicities

By understanding the process by which this occurs we can begin to developnew treatment strategies

Receptor; can respond to biochemical (ie TNF receptor/TNFα) or physical (integrins/cell deformation) stimuli

Singalling pathway; signalling pathways are a series of proteins that pass the signal along, usually by chemically altering the next molecule in the series-kinases; add a phosphate group-rybosylases; add a rybose group-ubiquitin ligases; add a ubiquitin

Its important to bear in mind that most of the chemical changes to molecule of A signalling pathway are reversible, ie, molecules can be used again.

Target molecule; it may be an enzyme, transcription factor or structural protein that is chemically altered by the signalling molecules, and thusly activated/deactivated

Components of a signalling pathway

Response; we can look at transcriptional responses (ie involvestranscription of particular gene or genes), translational responses (change inprotein levels), or functional responses (increase in enzyme activity, changein cell shape, production of certain product).

Transcriptional responses can be studied using standard PCR (1-6 genes),or micro-array analysis (100s of genes)

Translational responses can be studies using elisa, immunocytochemical,and western blotting techniques.

Functional responses can be studied using an endless array of assaysenzyme migration, deposition, etc etc.

Where to begin?

Example of functional assays

Calcium deposition and alkaline phosphatase staining in mesenchymalstem cells undergoing osteogenic differentiation

Studying the signalling pathway

Depending on what we already know about the nature of thestimulus/response we can either take an inside-out approach

Transcription factor – signalling pathway - receptor

Or an outside in approach

Receptor – signalling pathway – transcripton factor

The activation/deactivation of individual signalling pathway components canbe observed using techniques such as;

EMSA; transcription factors interaction with DNAIP; looks at protein/protein interaction ie. Ubiquitinisation, andphosphorylation

However it is often easier to inhibit or activate certain members of asignalling pathway in order to judge their importance to the phenomenon inquestion

RECEPTORS; receptor antagonists block receptor activation, while receptoragonists activate receptors; receptor antagonists/agonists are often usedclinically

For example pioglitazone, an agonist of PPARgamma

SIGNALLING MOLECULES; can be inhibited/activated using pharmacologicalor molecular inhibition/activation

Pharmacological inhibitors/activators are chemicals which inhibit or activatea molecule or family of molecules; they have the advantage that they aregenerally easy to use. However they are often toxic (to cells and scientists!)and relatively non-specific. Examples of pharmacological agents include;

Pertussis Toxin; inhibits G-protein signalling

Lithium Chloride; prevents degradation of b-catenin, therefore increasing itssignalling potential

Molecular inhibitors/activators; can be used to specificaly inhibit or activatea pathway

These can be tailored by the user for a specific molecule, and increasinglycan be purchased commercially

Anti-sense DNA oligos/plasmidssiRNADominant negative mutant plasmidsOverexpression plasmidsBlocking type antibodies

Studying cell signalling Case study # 1

1.BMP increases collagenase activityAddition of BMP/collagen formation assaysAntagonist/agonist studies

2.SMAD phosphorylationWestern blottingKinase inhibitorssiRNA against SMAD

3.SMAD/RUNX2 interactionImmunoprecipitation experiment

4. RUNX-2 activity Activity assay/reporter plasmid

Case study #2

1. Do Haemodynamic forcesregulate metallopeptidases inthe vascualar endothelium?

Used enzyme assays and mRNA analysisto monitor changes in cells that had beenexposed to cyclic strain

2. By which pathway does thisoccur?

Pharacological and molecular inhibition ofG-protein signalling pathways

4. Does this upregulation effectthe cells ability to processbradykinin/angiotensin

Looked at the cleavage of exogenouslyadded BK/Ang using HPLC, used avariety of inhibition strategies to inhibitenzymes

EP24.15/EP24.16 in the vascular endothelium

5. Does this change inpeptide processing effect cellfunction?

Looked at endothelial cell tubeformation with and without enzymeinhibition

3. Does cyclic strain cause achange in subcellular locization

Immunocytochemistry and confocalmicroscopy

Case study #3Effect of HIV infection on osteoblasts

1. Does HIV infection affectosteoblast function?

Treated HOBs with HIV proteins,observed changes in calciumdeposition and alkaline phosphatatseactivity with p55-gag and gp120

2. Do these changes inosteoblast function occuralongside changes insecreted signallingproteins?

Found changes in secreted RANKL,BMP2 and osteocalcin levels using elisakits

3. Are changes intranscription factor activitygoverning the response?

Found that the levels of RUNX-2 andPPAR-γ activity were altered bytreatment with p55 an gp120. usedcommercially available kits.