Slide Set 1: Techniques and Tools in Cell Bio

Cell Culture/ Tissue Culture

• Microscopy::

  • animal cell size- 10-30 micro m in size

• Rich Medium::

  • 9 essential aa

  • vitamins

  • peptide and GF (serum may be added)

  • neg charged solid surface to mimic extracellular matrix

• Primary Cell culture ::

  • cells prepared directly from tissues of an organism

  • display diff props of organs which they were harvested from

  • cannot be passaged as much

  • cell strain:: lineage of cells originating from one initial primary cell culture

• Transformed cells::

  • usually cells derived from tumor; cells that have undergone spontaneous genetic change

  • immortal cell line (not the same as cell strain)

  • grow to high densities

  • solid surface not required

  • con: may not accurately represent the og cells in tissue

• FACS::

  • cell sorter

  • can sort millions of cells in just a few hours

  • cells go through opening one by one, cells with no charge go in waste tube, cells that have florescence pass through charged plates and go to corresponding sides of the collection chamber

• Hybridomas::

  • used to produce monoclonal antibodies

  • monoclonal antibodies:: only can recognize a single epitope , used for covid treatment

  • to generate monoclonal antibodies:: inject antigen into animal, animal generates b cells to recognize antigen, myeloma cells fuse with b cells, transfer to to selective medium and find fused cells

Microscopy

• Magnification

  • objective lens:: glass lens close to sample

  • projection lens:: close to eye

• Resolution::

  • ability to distinguish btwn two obj

  • D= min distance btwn 2 distinguishable obj

  • N= refractive index

  • alpha= wavelength of angular aperture

  • Nsinalpha= NA

  • resolution is better= shorter wavelength= inc N= inc alpha

  • Bright field microscopy::

    • simple and inexpensive

    • no staining needed

    • live cell imaging

    • not clear for certain structures

    • there are ways to improve the limitations

  • Phase contrast Microscopy ::

    • modify the microscopy, certain components added to see edges of cells

    • thin layers of cells , live cells

  • DIC Microscopy ::

    • modified further , small details, 3D structures can be made

• Sample Preparation

  • Fix:: using diff chemicals to kill/section the cell, partially permeabilizes cells

  • embedding and sectioning- cut into small pieces to look directly at underneath the microscope

  • H & E staining::

    • hemotoxylin- stain nucleus, base, purple

    • eosin- stains cytoplasm pink, acid

• Fluorescence Microscopy

  • Fluorescent dyes/protiens- absorb light at shorter wavelength (excitation), emit light at longer wavelength (emission)

  • Fluorescence cell staining::

    • you can also co stain multiple colors to see different organelles/structures

  • Immunofluorescence Microscopy::

    • inject animal with primary antigen, harvest serum from animal that has antibodies in it, add 2nd fluorescent antibody to view

    • can only use dead cells

    • another way you can do this is by using GFP added to N term of protein to give it fluorescence without killing cell

  • Confocal Laser Scanning Microscopy (CLSM)::

    • focus on plane of thick specimen by rejecting out of focus light, helps improve clarity of images

• Advanced Fluorescence Microscopy

  • FRET::

    • goal is to measure the dist btwn entities, can also detect conformation changes

    • when CFP gets closer, YFP emmits color

  • FRAP::

    • looks at how fast a mem protein can move from one side of the membrane to the other

    • label with fluorescent antibody, bleach with laser, measure how quickly they move

  • Ion Sensitive Fluorescent Dyes::

    • can measure the amt of ions in a structure, can see how ions are distributed within cells

  • TIRF::

    • observe thin region of specimen, helps you look at v thin sections very close to the surface

  • Super resolution Microscopy::

    • we can break the resolution limit, take lots of pics and combine to improve resolution

• Electron Microscopy::

  • resolution is 2000x better than light microscope

  • TEM::

    • fix, embed, section (not live cells)

    • projects image on fluorescent screen

  • Immuno Electron Microscopy::

    • light fixation used, gold particles added to antibodies as electron dense marker bc they appears as dark spots under microscope

    • similar to light microscopy version

  • Scanning Electron Microscopy::

    • provide 3D image, scans surface and measures reflection

    • dead cells

    • reveals surface features of specimen

Perturbing Cellular Functions

• RNAi::

  • used to suppress the expression of genes in cells by blocking transcription in specific mRNAs or degradation of specific mRNAs

Studying Macromolecules

• Breaking up cells

  • separate organelles into pop of particles of diff density, shape, charge and size

  • Centrifugation::

    • more dense materials sink, lighter float

  • Magnetic Fractionation::

    • all the antibodies will bind to iron bc it is magnetic

  • Immunoaffinity Purification of organelles::

    • use antibodies to bind to organelles and purify them

  • Mass Spectrometer::

    • can tell us weight of particles

Slide set 2: Biomembrane structure

Biomembrane

• Biomembrane Functions::

  • selective permeability only certain things can cross membrane

  • compartmentalize organelles formed to localize biochemical reactions

  • scaffold for biochemical activities- certain proteins can receive signals on membrane

  • transporting solutes- certain mem proteins can transport things

  • signal transduction

  • energy transduction

  • intercellular interaction

  • have transmem, peripheral, and lipid surface anchor protein

• Lipids

  • amphiphilic :: have two diff properties, hydrophobic and hydrophilic

  • 3 classes

  • Phosphoglycerides::

    • include phospholipids

    • unsat- double bonds

      • cis vs trans

    • sat- no double bonds

    • PS and PI carry neg charges→ help determine charge of membrane

    • inc mem fluidity

  • sphingolipids::

    • some are phosphoglycolipids, some are not

    • sphingomyelin

    • dec mem fluidity

  • sterols::

    • not phosphoglycolipids

    • ring structure

  • cholesterol::

    • rigid and planar

    • hydrophobic tail inserts into membrane and polar group will extend beyond membrane

    • usually cholesterol dec fluidity, but at low temps it can inc fluidity

    • no cholesterol would mean the membrane would be too fluid, too permeable, the mem would disintegrate

  • 3 forms of phospholipid clusters::

    • liposome- has double layer membrane, used to deliver drugs

    • micelle- one layer membrane

    • bilayers

    • some conc can form structures, but it is v rare

  • Movement in the bilayer

    • transverse diffusion:: flip flop from one face to another, hard bc head group does not like to cross hydrophobic environment

    • lateral shift:: move across own face

    • flex :: turn around

    • flippase (ex to c) and floppase (c to ex) can aid in spontaneous flipping in the membrane, use ATP

  • Lipid rafts:: sphingolipid, cholesterol and certain proteins make up

• Exoplasmic vs cytoplasmic::

  • cytoplasmic face- faces to cytosol in cell/ matrix

  • exoplasmic face- faces exterior, or intermembrane space

  • both alternate, the cytosolic face will face the matrix, then the exoplasmic faces will both be facing the intermembrane space, then the cytoplasmic face will be facing the cytosol

• Liposomes as models

  • serve as models of biological membranes

  • can serve as impt tool in research, can carry antibodies, drugs, etc.

  • “synthetic vesicles”

Membrane Proteins

• Integral

  • crosses at least part of the bilayer

  • can be solubilized with detergents

    • detergents:: long hydrophobic and hydrophilic tails, can interact with a protein to keep it dissolved into the solution that it is in (SDS, Triton)

  • Transmembrane::

    • crosses bilayer from cytosolic to exoplasmic face

  • lipid anchor::

    • protein on the surface by lipid is conjugated to protein and lipid is in the bilayer

    • acylation- use fatty acids and conjugation on n terminal side

    • prenylation- conjugation happens on c terminus side

    • GPI anchor- has sugar and phosphate, conjugate to membrane on c terminus, face towards exoplasmic surface

      • synthesized in ER

• Peripheral::

  • less tightly associated, no lipid anchor, on surface of bilayer, can interact with negative head of lipids but cannot conjugate with them

Slide Set 3: Transmembrane Transport of ions and small molecules

Passive transport/simple diffusion

• Common features of passive transport::

  • everything is non polar and small

  • O2, CO2, N2, EtOH, water and urea

Mediated Transport

• Movement

  • electrochemical gradient- contains conc gradient and membrane potential (cyto more neg and exoplasmic side more positive)

• Facilitated Transport

  • uniporters::

    • transport single molecule down conc gradient

    • aquaporins and glucose transporters

    • fast, reversible, specific, conformational change

    • GLUT1- glucose transporter expressed on plasma membrane of RBCs and endothelium or BB barrier, can pump one glucose at a time

    • aquaporin- water transport pore, transports water across membrane for water balance, arrangement inside porin prevents protons from entering

    • aquaporin 2- absence of it leads to diabetes, excretion of large vols of dilute urine

  • Ion channels::

    • form hydrophilic tube that ions can move down conc gradient

    • facilitated diffusion

    • faster than uniporters

    • Na/K- inside cell is negative, charge accumulates on sides. Both go from high to low conc. K moved to inside cell, Na to outside of cell , the K channel is an ion channel, Na/K pump is ATPase

    • patch clamping - can use small needle to clamp onto membrane and suck part of the membrane with the channel in, we can measure current flow with this technique

• Active transport

  • ATP powered pumps::

    • move molecule from low conc to high conc using ATP hydrolysis

    • p class pumps

      • in plasma membrane of plants, fungi (H+ pump), and eukaryotes (Na+/K+ pump and Ca pump)

    • V class proton pumps

      • seen in many mems, almost only transport protons, couple ATP hydrolysis to transport protons against conc gradient

    • F class proton pumps

      • seen in bacterial plasma mems, almost only transport protons, utilize energy in proton concentration or voltage gradient to synthesize ATP

    • ABC superfam

      • seen in bacterial and mammalian plasma mems, have 2 transmem domains and 2 cytosolic domains that couple ATP hydrolysis to solute mvmt, switch conformation and move from one side of mem to other

  • symporters::

    • move one molecule from low conc to high conc and another from high to low, molecules moved to same side

    • conformational change

    • Na/Glucose symporter

  • antiporters::

    • move one molecule from low conc to high conc and another from high to low, opp directions

    • In systemic capps- Cl/HCO3 antiporter- CO2 enters cell via passive diffusion and will react to form HCO3, H2O also is involved and H is used to bind to histadine and will form hemoglobin O2 molecule. HCO3 out, Cl in, HB out

    • ^^ can be backwards in pulmonary capps- HCO3 in, Cl out, HB in so that CO2 can leave

• Bulk movement

  • vesicular transport::

    • Pinocytosis- small nutrients taken up by the cell, fluid droplets

    • Phagocytosis- large obj taken up by cell, bacteria

    • exocytosis- secretion of obj out of cell

  • transcellular transport::

    • glucose from lumen to blood

      • Na K ATPase- uses ATP to move Na and K from low to high conc

      • Na Glucose Symporter- uses Na and mem potential to pump glucose and Na from low to high conc

      • GLUT2- glucose uniporter

    • acidification of stomach lumen

      • combined action of 4 diff transport proteins acidifies stomach lumen while maintaining nuetral pH of cytosol

      • H K ATPase

      • K channel

      • Cl HCO3 antiporter

      • Cl channel

Slide set 4: Moving Proteins into Membranes and Organelles

Eukaryotic Protein Trafficking

• Ribsomes

  • mem bound :: attached to cytoplasmic face of ER, synthesize secretory proteins

  • free ribosomes:: free in cytosol, synthesizes non secretory proteins

  • polyribosome :: mRNA molecule to which ribosomes are attached and engaged in protein synthesis

• Secretory pathway (ER ribosomes)

  • secreted proteins, many plasma membrane proteins, resident proteins of secretory pathway

  • Steps of secretory pathway ::

    • import into ER

    • folding and glycosylation in ER

    • vesicular transport from ER to golgi

    • modifications in golgi

    • vesicular trafficking to final destination: plasma membrane, lysosome, endosome

  • Signal hypothesis::

    • signal sequence at n term of proteins destined for import into ER

    • signal seq bound by signal recognition particle (SRP)

    • SRP binds to GTP which is bound to the translocon

    • translocon translocates protein into ER

    • GTP released

    • signal peptidase cleaves off signal seq

  • Co translational translocation::

    • signal peptidase cleaves signal seq

    • proteins cont into lumen of ER

    • protein folds in Er, ribosomes detaches and floats off to being process again

  • Post translation translocation::

    • no ribosomes is bound to protein, peptide chain goes through translocon

    • BiP protein binds to Sec63 complex and hydrolyzes ATP

    • ADP cont to bind to protein along the chain and the ADP remade into ATP

  • Microsomes::

    • homogenized rough ER and smooth ER, makes vesicle like structure from pieces of ER

    • can show us that translation and translocation can occur simultaneously

  • SRP structure::

    • methionine residues that bind to signal seq on P54

  • ER membrane proteins

    • Type 1 ER mem proteins::

      • n term faces to lumen, c term to cytosol

      • translate until there is a stop transport anchor seq- translocation stops, translating continues

      • protein synthesis cont, but translocon is closed and the synthesis continues just in the cytosol

      • ribosome leaves, protein seq left embedded in mem

    • Type 2 ER mem proteins ::

      • n term faces cytosol instead

      • signal anchor seq- stops translocation into cytosol, translocation into lumen commences and transcription is cont

    • Type 3 ER mem proteins ::

      • n term faces lumen, c term cytosol

      • protein synthesized through translocon until signal anchor seq

      • translocon closes, transcription cont in cytosol

      • basically same as type 1 except C term tail is long here instead of n term tail being long

    • Type 4 ER mem proteins ::

      • multiple transmembrane domains

      • 4A- n term faces cytosol

      • 4B- n term faces lumen

    • GPI anchor protein ::

      • hydrophobic c term

      • ATP binds to c term

      • ATP binds to GET proteins

      • ATP hydrolyzes to transfer c term into ER mem

      • ATP synthesized

  • Hydropathy::

    • how to tell if proteins are transmembrane proteins

    • give individuals diff numbers, hydrophilic gets neg numbers, hydrophobic gets positive numbers

  • Resident Er proteins assist in Folding and Glycosylation in the ER

    • Binding protein (BiP)::

      • assists in post translational translocation

    • Protein Disulfide Isomerase (PDI)::

      • catalyzes the formation and rearragement of disulfide bonds (key for proper folding, function, and stability )

    • Glycosyltransferases::

      • N linked

        • carb chains attached to the amide nitrogen of asparagine called n linked

        • variable

        • involved in glycosylation

        • there is a signal for addition, quality control, folding of protein

      • O linked

        • carb chains attached to hydroxyl in serine and theronine residues

• Organellar Targeting (Free ribosomes)::

  • nuclear proteins, mitochondrial proteins, chloroplast proteins, peroxisomal proteins, some plasma mem proteins

  • protein residents of other organells are directly targeted to those organelles independent of secretory pathway functions

  • most translated on free ribosomes

  • may or may not fold in cytosol

  • post translationally targeted into organelle

  • Mitochondria

    • Mito Encoded::

      • transcribed in the mito

      • translated on intro mito ribosomes

    • Nuclear Encoded::

      • most mito proteins

      • transcribed in nucleus

      • translated in cyto

      • imported into mito

    • Mito Import machinery::

      • Requires:

      • Import receptor- reads mito targeting signal, delivers to translocon

      • Translocon of outer mem- associates with import receptor, delivers protein to 2nd translocon

      • Translocon of Inner membrane- aligned with TOM

      • Matrix in Hsc70 (acts like BiP)- aids in net translocation

    • Path A and B

      • contain an N term matrix targeting seq that is recognized by the Tom 20/22 import receptor in outer mem. Only diff is that the entire precursor protein enters the matrix and is then redirected to the inner mem in path b

    • Path C

      • contain internal seq that are recognized by the Tom 70/22 import receptor , Tim 22/54 is also used

  • Peroxisome::

    • nuclear encoded

    • post translationally imported into peroxisome

    • imported as folded proteins

    • two targeting seq, PTS1 and PTS2

    • Pex5 is receptor, binds to Pex14, transfers to Pex12/2, then Pex5 releases

  • Nucleus::

    • all proteins translated in cytosol

    • folded proteins transported

    • nuclear localization signal for nuclear proteins, near c term

    • nuclear pore complex- spans both bilayers, huge

    • nuclear import- occurs via diffusion

    • nuclear export- driven by Ran GTPase

    • Ran Independent nuclear import also exists