153 Cards in this Set
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In general- but not always!-
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one gene codes for one protein.
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Genes can be....while proteins can be
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· Genes can be spliced alternatively, proteins can be cleaved into multiple active species
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Proteins fold into specific shapes to make:
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—Enzymes, which carry out chemical reactions
– Structural proteins, which provide organization
– Regulatory molecules, which provide information
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Changes in DNA produce
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heritable changes in protein structure and function.
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Proteins are the
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workhorses of cells- they do most everything cells do.
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We can “read” the sequences to predict the complement of proteins of many organisms
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Compliment of proteins=the PROTEOME
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How many proteins does it take to make a cell?
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– Prokaryote minimum: 477 in Mycoplasma (parasite). Typically:
1000-4000
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Eukaryote: Simple single-celled: 6300;
Complex multicellular: 30,000
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If genome is known & all cells of an organism contain same DNA, is cell's behavior predictable?
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NO, because remember, a cell, whether single-celled organism or a cell in a multicellular organism, does not express all of its genes all of the time.
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Differential Gene Expression
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in any given cell, some genes are turned on, some are turned off. some are expressed at high levels, some low.
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Transcriptome
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the genes being transcribed
Depending on which genes are being transcribed and translated, a cell will express a particular suite of proteins
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Signal Transduction
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One of the central goals of cell biology is to understand how all those proteins interact with themselves, and with other cellular components, to carry out cell functions or to respond to changes inside or outside of the cell
Sensing these changes or stimuli, communicating them through…
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Various signal transduction responses
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some cellular responses may act through the existing proteins of a cell. some may lead to changes in gene expression that change the suite of proteins in the cell.
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Transcription factors
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are proteins that bind DNA and can activate or repress the transcription of their genes, and they are often targets of signal transduction pathways.
Since transcription factors can regulate the expression of other transcription factors, cells can develop complex gene regulatory netw…
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Transcriptional changes are....
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often TEMPORARY and REVERSIBLE in response to stimuli.
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Epigenetics
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Heritable changes in gene activity that are not caused by changes in the DNA sequence.
A change in the gene expression, (on or off) but leaving the DNA sequence untouched.==METHYLATION
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2 Basic types of cells on earth
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Prokaryotes lack a nucleus and internal membranes
Eukaryotes have a nucleus and an extensive internal membrane system.
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3 types of eukaryotic cells
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Protists, animals and plants
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Structure of a Prokaryotic Cell (6)
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Plasma membrane: Lipid bilayer-boundary of cell that allows it to create an internal environment different from the external environment (special systems exist to transport both small and large molecules cross this barrier.) Cytoplasm: the soluble internal contents of the cell
Nucleoid: …
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Basic structure of Eurkaryotic cell (animal or protist)
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In addition to prokaryotic structure add: Internal Membranes and organelles– spherical vesicles, sheets and tubules perform special functions Cytoskeleton– networks of filaments inside cells – provides structural and organizational framework of cells – provides oriented railro…
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Plants and Algae=eukaryotic cells with a lil something extra
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in addition to the components that animal and protist cells have, plants and algae also have: \
Chloroplasts=make ATP from sunlight
Cell walls:
Rigid cellulose walls (extracellular matrix),
Define shape of cells and tissues
For protection & strength;counter osmotic turgor pressure
…
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major difference between plant and animal cells
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Plant cells have chloroplasts and cell walls.....animal cells DO NOT
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Differences between prokaryotes and eukaryotes
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Eukaryotes have a nucleus
Eukaryotes have organells
Eukaryotes of a cytoskeleton
Eukaryotes have internal membranes and membrane trafficking
Prokaryotes have flagellum
BOTH HAVE PLASMA MEMBRANE
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Common ancestor rule
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Tree of life, all life share common ancestor + DNA and RNA sequences are conserved across many organisms and have changed over time
All organisms have ribosomes which means all organisms have RNA?
Just because we share a common ancestor doesn't mean that we only came from one primit…
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How did first cells arise?
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there was a soup of organic molecules that was created abioticall(by non lving processes. Earlier versions of Cell Theory thought that living cells could arise from non-living material
c. RNA can both code for genetic information and act as a catalyst.
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presumed Evolution of cells
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Prokaryotes evolved first. They were simpler cells.
Eukaryotes evolved from prokaryotes, via processes involving internalization and eventual symbiosis,
-----More complex cells need more genes, so the nucleus may have evolved to keep track of the increasingly large genome.
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Proposed evolution of eukarotes
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1. Heterotroph ate a aerobic prokaryote = symbiosis emerged and formed as a mitochondria
3. Nucleus made from invagination which formed because we needed more surface area to store more DNA
Box. Could also have formed from a cell that ate another cell that was more effective at stor…
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How did behaviors of the cells arise
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All behaviors of any cell arise solely from the interactions of proteins and lipids and nucleic acids.
Protists are most complex of single cell organisms
Protists do not have brains or nervous systems, they only function from interactions with different stimuli or the chemicals list…
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Light microscopes are limited in (3)
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Magnification;
Contrast:
Resolving power:
--this limits what can be detected with them
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Magnification
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Magnification; the amount you blow up the initial image, dependent on the lenses you use
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Contrast
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Contrast: the difference in intensity between an object and its background. to enhance contrast, exploit changes in the phase of light (brightness) or stain (color) the object to make it darker
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Resolution
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Resolving power: how far apart two objects have to be to be seen as two separate objects.
Dependent upon the properties of light and how it interacts with the specimen.
Determined by: WAVELENGTH OF LIGHT--shorter wavelength=better resolution; and by the width of the light cone the objec…
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Fluorescence
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The absorbance of invisible, UV light by a compound and the subsequent release by that
compound of some of the energy as longer, visible light wavelengths is known as
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short, longer
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In a fluorescence microscope, ______-wavelength incident light is absorbed by the specimen and reemitted at a _______ wavelength.
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fluorescence microscopy
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In which light microscope technique does a molecule absorb energy from light and glow with
a bright color or colors against a dark background? The method is often used to localize specific
molecules within a cell.
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fluorophores
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A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation.
They are linked to various chemicals or biological molecules so as to label specific structures Green=protein Yellow=antibody Red=protein Purple=antibody Blue=DNA binding dye
Flurophores …
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Why is the contrast from the epifluorescence microscope excellent?
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Because specific molecules glow brightly against a dark background.
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Looking under a transmitted light microscope
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An object appearing 200nm or smaller will appear to be 200 nm regardless of its actual size
If you see a 200 nm spot in a microscope, it could be:
ONE 200nm object
TWO 100nm Objects close together
FOUR 50nm objects close together
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Immunocytochemistry
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Uses antibodies to visualize proteins in cells
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What are antibodies
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Antibodies are proteins that bind to specific proteins
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How do you make an antibody?
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To make an antibody, you obtain pure protein of interest and inject it into an animal. the animal recognizes it as foreign and mounts an immune response. the antibody that results will recognize a specific 8-12 amino acid sequence. (EPITOPE)
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How does one use antibodies to label cellular structures?
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You must fix and permeablilze the cell.
Add the antibody to the fixed permeabilized cell and allow it to bind its target
Use a fluorescent "secondary antibody" (an antibody against antibodies) to bind to the primary antibody if the primary is not directly labeled.
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What does it mean to fix and permeabilize the cell?
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Fix: add chemicals that cross-link everything in the cell to nearby molecules.
Permeabilize: add a detergent to remove some of the membrane so antibodies can get in.
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35. Antibodies can be used to
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a. visualize proteins in fixed and permeabilized cells
b. identify specific proteins on an immunoblot (western blot)
c. precipitate proteins from cellular extracts
d. identify and isolate organelle fractions
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Western Blotting (immunoblotting)
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Is the name of the procedure in which proteins separated on a polyacrylamide gel are transferred with the application of a current to a nitrocellulose filter placed against the gel and subsequently identified by their interaction with specific antibodies?
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How are monoclonal antibodies made?
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By cloning an antibody-producing cell, and thus recognize a single epitope. All the antibody molecules produced are identical
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What are polyclonal antisera?
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They are a mixture of different antibodies produced by the host animals immune response against various epitopes of the target protein.
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What are fluorescent proteins?
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Fluorescent proteins are genetically encoded fluorescence markers that can be fused to proteins of interest at the DNA sequence level. They allow LIVE IMAGING
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Transfection can be made to be TISSUE SPECIFIC
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Transfect = infect + trans (across)
Formation of the plasmid to create a Green Fluorescent Protein, can be made in such a way to select for a a specific tissue to make it fluorescent across all of it's tissue
Introduce cDNA that allows for selection of of proteins that will fluores…
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Transfection
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A process used to infect a cell with foreign DNA; to cause a foreign protein to be expressed in a cell.
You can transfect mutant cells that alter normal function.
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Adding to GFP-tagged versions of molecules present in cell, U can express (either tagged or not):
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Molecules that arent normally present in a cell
Mutant molecules that are constitutively active- i.e. active all the time
Mutant molecules that are dominant negative- i.e. don't function right and block the function of the cell's own version of the molecule.
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What are two ways that cells can be transfected?
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Cells can be transfected transiently or stably
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Name an example of what results from a cell that undergoes a stable transfection?
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A powerful variant of this basic method results in the stable transfection of an entire organism with DNA, yielding a transgenic animal. Protein expression can be general, in all tissues, or under the control of a tissue-specific promoter (brain, heart, etc.). Proteins can also be knocked…
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siRNA
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Small interfering RNA
Today, there are new technologies that allow specific proteins to be knocked out in cells.
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What are the methods to deblur images to remove out of focus light?
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Laser scanning confocal microscopy use pinholes to deblur since out of focus light is blocked, generating an "optical selection"
Digital deconvolution uses computational methods to deblur
Blurring is due to the parts of the cell above and below the focal plane fluorescing
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How is electron microscopy useful?
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Another means of getting around the resolution limits of light.
EM lenses aren't as high quality as optical ones, but still much more powerful than optical light microscopes
Electron beams have a shorter wavelength and thus a higher resolution Wavelength is 0.004 nm (as opposed to 400-5…
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Transmission electron microscope (TEM)-
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Images electrons that pass through a thin specimen
Actual resolution = 0.1-2 nm (100-1000x better than light microscope)
If lenses were as good as optical ones, resolution would be 0.002 nm
(100,000x better than light) but NA of magnetic lenses is much worse
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Scanning Electron Microscope
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Images electrons scattered by an intact object. Depth of focus gives images a three-dimensional quality.
Resolution of SEM is about 5 nm
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How is an electron microscope similar to a light microscope?
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The transmission electron microscope is just like the light microscope except with electrons.
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Rules for Electron microscopy
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EM must be done in vacuum for electron gun to work – Can’t have wet samples
Dry tissue doesn't have enough density to scatter electrons so must replace it w/ something dense– Bind metals like uranium, lead, or tungston to membranes & proteins
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Procedure for preparing a Transmission Electron microscopy
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Fix Tissue (glutaraldehyde-proteins; osmium tetroxide-lipids)
Stain with uranium, lead etc. or "metal shadow" after sectioning
Dehydrate and embed with plastic
Cut thin slices (sections) (0.02-0.1μm thick)- sample
must be thin otherwise electrons don’t get through
What U see = the sc…
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Two modes of EM
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TEM (does thins slices) has higher resolution than scanning EM
Scanning (Scans surface) has worse resolution
EM can't be done on living cells cause the process to view them through an EM kills cells
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Antibodies, in a way similar to immunofluroescence
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allow for identification of cell structures through them tagging to specific structures
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How can you label structure in electron microscopy?Immuno-electron microscopy
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You can’t see antibodies in the EM, but you can attach dense particles to antibodies to make them visible in the EM
Allows you to visualize the localization of specific proteins in the EM
Very difficult technique!
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Scanning Electron Microscope
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Used to look at surfaces of structures
Samples are fixed, passed through series of alcohols, and dried. Surface of sample is coated with a layer of metal.
The electron beam is scanned across the surface and the reflection of electrons at each point measured.
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Compare Light, TEM and SEM microscopy
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b) phase contrast light microscopy - hair cilia in ear
a) SEM - electrons are scattered a thin layer of metal, allows for a 3d image
c) TEM - Thin section appearance, the cilia aren't broken off, but rather the tilt of the image shown seems to show the cilia not being fully connecte…
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 A great deal of what we know about cells comes from
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Biochemical experiments conducted with organelles isolated and purified by gradient centrifugation
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Differential centrifugation -
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Take out each pellet as you work to get to the next group of cellular structures to study
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A key difference between prokaryotic and eukaryotic cells is in
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the elaboration of internal membranes
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Each membrane-bound compartment in a eukaryotic cell has
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ts own unique set of soluble proteins
AND
its own unique set of membrane proteins.
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Membranes perform several important cellular functions
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1. They separate one compartment from another -Membranes are selectively permeable barriers across which solutes are transported
2. They provide a scaffold for biochemical activities -One key example is energy transduction in mitochondria and chloroplasts
3. Plasma membranes mediate som…
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What are membranes?
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Membranes are fluid lipid bilayers studded with proteins and containing areas of differing composition called rafts that float around like icebergs on the ocean
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Three classes of membrane lipids
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Phospholipids, Glycolipids and Sterols
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Phospholipids
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• All have a phosphate linkage to a “head” group, and 2 fatty acid chain “tails”.
a class of membrane lipids
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Two types of Phospholipids
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• Phosphoglycerides
• Sphingomyelin
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Describe Phosphoglycerides
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– Major component of most membranes
– Consist of two fatty acids linked to glycerol, with differing chemical groups added to glycerol phosphate in the head group.
– One fatty acid chain is saturated, one unsaturated
– Many different types with different structures
1/2 phospholi…
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Describe Sphingomyelin
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– Sphingosine amino group instead of glycerol linkage to phosphate in head
– Two saturated fatty acid chains
2/2 phospholipids
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Glycolipids
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a class of membrane lipids
– Sphingosine amino group links to sugars instead of phosphate in head
– Have two saturated fatty acid chains
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Sterols
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a class of membrane lipids
Sterols are amphipathic, four-ring hydrocarbons. Cholesterol can increase or decrease membrane fluidity depending on conditions.
• Saturated fatty acid chains give rise to thicker and less fluid bilayers
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Lipids in water can form two types of structures
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Micelles and bilayers
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Need to get hydrophobic tails of phospholipids away from water- 2 common ways of doing this
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Micelles-small sphere with tails pointed in
Bilayers-Two layers of lipids with tails pointed toward each other
Which structure is formed is dependent on the type (chemistry-charge on head, tail length, tail shape) and concentration of the lipid
Biological phospholipids can form bilayer…
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Properties of Lipid Bilayers
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Bilayers close upon themselves to make a continuous surface interacting with water.
No “edges” are left exposed to prevent water interacting with hydrophobic tails
Membranes try to reseal if broken or punctured
– A cell will die if the seal does not reform fast enough
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Phospholipid bilayers are
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Phopholipid bilayers are self-forming, and self-repairing (driven strictly by energetically favorable formation) - They can spontaneously heal
Proteins embedded within the lipid membrane allow for different functions
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Biological Membrane Composition
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Different cellular membranes are composed of different amounts of lipids and cholesterol, and have very different protein composition
The differences in composition relate to differences in function
Each biological membrane has different properties based upon the different molecules use…
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Lipid composition affects
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bilayer thickness and membrane curvature
cholesterol straightens the PC to make it longer
Seeing this under a light microscope you would see it as 200 nm even though it's actually composed of compounds at a size of nm's
Cholesterol changes membrane fluidity
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Membranes are dynamic structures: summary of movements of phospholipids
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Plasma membrane composition is asymmetrical, flip-flopping is energetically unfavorable and the least likely, flexion is the most common, with lateral shifts being inbetween
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Using Microscopy to determine the lateral mobility of lipids in the plane of the membrane
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Fluorescence recovery after photobleaching (FRAP)
– Labelphopholipids with a fluorescent probe
– Shineabrightlaseron a small spot of membrane to bleach it
– Measurehowlongit takes for fluorescent molecules to diffuse into the black region until it is as bright as the rest of the mem…
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FRAP Fluorescence recovery after photobleaching (FRAP)
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Incorporate fluorescently tagged cells into bilayer
Shine laser beam to burn out/bleach area hit w/ layer (done to create dark spot)
Then phospholipids move around & eventually dark spot isn't dark anymore & fluorescence recovers.
Won't recover if the lipids cant move
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The two different leaflets of the membrane have different compositions
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– The lipids are synthesized and inserted into one or the other faces of the bilayer
– Membrane proteins (flipases) flip-flop the lipids back to their normal sides to maintain the asymmetry!
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Membranes aren't uniform seas of phospholipids w/ proteins floating in them
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the're asymmetric!
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Lipids are not distributed randomly in the plane of the membane
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– Some lipids, especially sphingolipids, like to cluster relative to other membrane lipids
– This creates areas with locally high concentrations of certain lipids and proteins. These are called Lipid Rafts
Certain areas have different lipid compositions, thus they may form lipid raf…
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What is the other major component of membranes?
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PROTEINS
Lipids serve to create a barrier, proteins allow selective permeability, transmit signals, conduct biochemical reactions, etc.
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The Central Dogma
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DNA synthesis (replication)
RNA synthesis (transcription)
Protein synthesis (translation)
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Proteins are the basic machinery of cells. They serve three basic roles
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enzymatic
structural
regulatory
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There is a hierarchy of protein structure
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Primary Structure-the sequence of amino acids, determined directly from the RNA sequence Secondary Structure- local shape (beta sheet, alpha helix)
Tertiary Structure- 3D shape of whole protein (3d shape of single polypeptide chain)
Quaternary Structure- multi-subunit assemblies (severa…
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Primary Structure
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-ALA-GLU-VAL-THR-ASP-PRO-GLY-
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Secondary Structure
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Alpha helix and Beta pleated sheet
Hydrogen bonds are main contributor to secondary structure.
Hydrogen bonding pattern is determined by primary amino acid sequence.
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Tertiary Structure
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Tertiary structure is determined by:
– Hydrogen bonds
– Hydrophobic interactions
– Ionic interactions
– Polar interactions
– Van der Waals forces
– Covalent-disulfide bonds
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How can Protein Folding occur?
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Co-Translationally
Many ways to fold a protein, only one is right
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Protein Folding
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Sometimes, N-terminus hydrophobic domain should pair with one at the C-terminus.
B/C protein synthesis occurs over time, the right C-terminus may not even exist for a while, creating a situation where the N-terminus pairs incorrectly.
Some proteins fold properly on their own, others…
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Protein Folding Chaperones and Chaperonins
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Chaperones and Chaperonins are proteins that bind to proteins during or after synthesis and help them fold properly.
Some proteins need even more help from protein complexes called chaperonins
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HSP Family Proteins
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HSP Family Proteins are the major chaperones in all organisms
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Some proteins need even more help from protein complexes called chaperonins
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Some proteins need even more help from protein complexes called chaperonins
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Disulfide bonds stabilize protein structures in oxidizing environments
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Cytoplasm is not an oxidizing environment, so disulfide bonds are uncommon in cytoplasmic proteins
Inside the ER or outside of the cell are oxidizing environments - Inside the cytoplasm are not
Cytosolic proteins do not have disulfide bonds
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Proteins are comprised of Functional domains
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A combination of helices and sheets with turns and connecting regions can fold into a functional domain that acts as a unit but is still only part of a protein. Functions can include things like:
– ATP binding sites (myosin motor)
– Ca binding sites
– Enzyme activity of a particul…
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A protein normally consists of more than 1 domain. Each domain performs a specific fxn
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Evolutionarily, new proteins can be formed by putting together new combos of domains (domain shuffling)
Some of same domains get used over & over by diff proteins in different cells
It is increasingly possible to analyze primary sequences & deduce the FXN of protein by analyzing domain …
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Mammalian PLC is composed of
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domains that are found in other proteins
Phospholipid hydrolase - Hydrolyzes phospholipids
Blue: catalytic
Green: EF-hand (Ca binding) Yellow: pleckstrin homology (PH) domain (lipid binding)
Red: C2 domain (Ca and PS binding)
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Most membrane proteins and secreted proteins are
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glycosylated via processing in the ER and Golgi apparatus
Glycoproteins are oriented so that the carbohydrate chains are in the extracellular domain
Oligosaccharides are used as tags to mark the state of protein folding.
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How Quaternary structure is formed?
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Formed by interaction between 2 or more subunits to form a complex. 2 subunits=dimer, 3= trimer, etc.
If the complex is composed of two identical subunits, it is a homodimer. If the two subunits are different polypeptides, it is a heterodimer.
Protein complexes can be stable, or can…
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Quaternary structure is determined by:
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– Hydrogen bonds
– Hydrophobic interactions
– Ionic interactions
– Polar interactions
– Van der Waals
– Covalent-disulfide bonds
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What determines the way proteins bind to other proteins or DNA?
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Affinity = number of bonds that are formed formed between two interacting species that determines the stability. a and d have many bonds with one another, thus they have a high affinity
AFFINITY
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What does affinity control?
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Affinity controls what fraction of a molecule will be bound as the concentration of it's binding partner is varied
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Kd Trends size of Kd to affinity levels
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The smaller the Kd, the higher the affinity- i.e A is more likely to be bound.
The larger the Kd, the lower the affinity, the less likely A is to be bound
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What is Kon dependent on?
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Kon depends on the rate of diffusion, the size of the molecules, and whether there is a favored orientation required for binding.
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What is Koff dependent on
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Koff is what really determines the AFFINITY of the interaction. It depends on the sum of the
forces (ionic bonds, van der waals interactions, hydrophobic interactions) that will
hold A and B together. The smaller the Koff, the smaller the Kd. That means that complexes will stay bound lo…
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What does affinity determine?
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Affinity determines the 50% point of a reaction, less than that and it can be said to not be bound, higher than 50%, and it can be said to be bound
Affinity = how long things tend to stay bound after they interact
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Three Classes of Membrane Proteins
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Integral membrane proteins
Lipid anchored proteins
Peripheral membrane proteins
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Integral membrane proteins
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Tightly associated with the lipid bilayer.
Interact directly with lipid portion
Transmembrane- span the bilayer one or more times.
Others associate with only one leaflet
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Lipid anchored proteins
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Covalent addition of a lipid to a protein targets the
lipoprotein to the membrane.
Fatty acids are added to attach proteins to the inner leaflet Glycophosphoinositol (GPI) added to attach proteins to outer leaflet.
Some lipid anchored proteins can cycle between membrane- bound and solu…
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Peripheral membrane proteins
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Attached to the membrane via interactions with other membrane proteins, not lipids
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Integral Membrane Proteins are integral because
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Integral because a portion of structure is embedded within the bilayer
1. Single transmembrane domain protein
2. Triple pass transmembrane domain protein
3. Beta barrel protein
4.Interacts with only one leaflet
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How do you show a protein is associated with the membrane?
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•Immunofluorescence or immuno-EM to localize protein to membrane
•Purify the membrane and determine which proteins are present\
•Purify protein & show that association w/ membrane lipids is required for its function
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Purify the membrane and determine which proteins are present: HOW?
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–Break cells by homogenization
–Membranes have diff. density than other molecules allowing them to be separated via sucrose gradient centrifugation
–Special detergents (trypsin) used to dissolve membrane but keep membrane protein active
–Diff membranes of cell have diff compositions & …
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What do Proteases do?
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Proteases cleave accessible protein regions and can be used to deduce topology of a protein in the membrane (modified from Karp 4-17)
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What is the structure of the transmembrane region of proteins?
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Hydrophobic/ amphipathic alpha helix or amphipathic beta sheet
• If alpha helix-based, can span one or more times
– Single Pass Transmembrane- crosses the bilayer once
--Multipass Transmembrane- crosses 2 or more times
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What is the structure of the transmembrane region of proteins?
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• The length of an alpha helix needed to cross the membrane is about 20-30 amino acids. Remember though that different lipid composition gives rise to different bilayer thickness, so the length of a membrane protein’s hydrophobic alpha helix will influence what kind of lipid composition …
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A multipass protein example
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bacteriorhodopsin (10-37)
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Water-filled pores through the membrane can be made from amphipathic helices
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Even number of hydrophobic --> Amino terminus and Carboxy terminus on the same side
Protein with Odd number of transmembrane helices --> amino and carboxy are on the opposite side of the membranes.
The "inside" of the helices are hydrophilic to allow for a pore to let water in.
Four p…
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Beta sheets can also interact with membranes
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• The R groups from the sheet are organized so that the non-polar are on one side and polar on the other
• The sheet is rolled into a tube (Beta-barrel)
• The hydrophobic are out toward the bilayer
• The polar are inside
• Forms pore through membrane that is a hydrophilic environ…
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The Fluid Mosaic Model of Membranes
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The lipid bilayer is a flexible 2 dimensional fluid sheet
Membrane proteins “float” in this sheet
Proteins can move laterally in the plane of the membrane
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FMM of Membranes-Proteins can move laterally in plane of membrane, but...
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– A protein cannot easily leave the membrane once inserted.
Too much energy is required to tear the hydrophobic region out of the hydrophobic bilayer.
– The topology of a protein cannot easily change once inserted in the membrane.
If it is made with 7 transmembrane regions, it will …
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Many proteins are constrained & cannot move freely w/in the membrane
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• Not all membrane proteins=mobile! • Some membrane proteins= restricted in their location to particular region of membrane – membrane domain • In single cell, some proteins maybe evenly distributed while others=restricted • The same protein may be restricted in one type of membr…
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Method 1 for measuring protein mobility: cell fusion
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• Label proteins of 1 cell w/ red dye • Label those of a 2nd cell w/ green dye • Fuse membrane of two cells to form heterokaryon
Note-cell membranes normally don't fuse. U have to force it happen
• Watch what happens to the two dyes
• Result- over time they become mixed
Heter…
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Method 2 for measuring protein mobility: FRAP
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FRAP of fluorescently labeled membrane protein
Fusion protein/ chimera of CD2 to YFP
FRAP, shined a laser on the cell, and then saw when the dark spot went away
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Method 3 for measuring protein mobility: single particle tracking
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• Particle Tracking: Label one or a few of the molecule interested in - Use a fluorophore (quantum dot - very bright) sparsely label a few per cell and then look over the cell and follow the light emitted over time
• The 200nm spot can be traced out to find where the fluorescence was s…
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Lipid rafts:
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• Raft components include cholesterol, sphingolipids, and proteins. Rafts tend to accumulate different proteins than the non-raft areas.
• Bottom is a cartoon interpreting the top view, cholesterol in blue, the bunching of it likely causes the Lipid rafts red it different lipid composi…
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Lipid rafts: Longer Helix
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The Longer helix (it is a protein) has its alpha-helix exposed to a watery envrions, the helix is hydrophobic, thus the interaction is high energy since it's bombarded by molecules that it doesn't "like", thus the alpha helix will be driven to a thicker section of the bilayer
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2 ways to measure mobility of cells:
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FRAP and Particle tracking
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FRAP
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FRAP: Label as many molecules as possible to make sure the mobility is checked throughly
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Particle Tracking
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Particle Tracking: Checks for affinity within a complex of a molecule by attaching a fluoresence molecule to an antibody connected to the protein in question. Want to label as little or relatively few molecules
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Permeability properties of the plasma membrane!
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• Plasma membrane functions as a selective permeability barrier!
• Molecules diffuse across membrane at different rates!
• Some able to move through lipid bilayer more easily than others based upon their chemical properties!
• For those that cannot easily cross membrane there are t…
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Small uncharged molecules can move across the lipid bilayer by passive diffusion!
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• Passive diffusion=when molecules diffuse directly through lipid bilayer- no protein Involvement! • Some molecules diffuse through easily! – Small, uncharged, non-polar! – Gasses (O2, CO2) cross easily! – H20 crosses, but less well!
• Small but charged cross very slowly: Ca2+, K+…
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Rate of movement with passive diffusion is dependent on...
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• Rate of movement is directly related to solubility of molecule in lipid bilayer (partition coefficient) !
– The more soluble a molecule is in the lipid bilayer, the faster it will diffuse across!
Water can pass through the hydrophobic tails of the bilayer just because of high amou…
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Membrane Transport Proteins!
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The solution to the limited permeability of the membrane for some substances is for cells is to have transport proteins in their membranes. These proteins are enzymes that catalyze movement of specific substances across the membrane.
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Passive Transport!
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– Passive transporters allow net movement down a chemical, electrical or electrochemical concentration gradient; they require no energy beyond thermal motion!
• Ion Channels!
Carrier proteins (facilitated diffusion)
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• Active Transport!
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– Active transporters can move molecules against a chemical, electrical or electrochemical gradient; they require extra energy input for transport!
• ATP-dependent pumps!
• Symporters (a.k.a. contransporters)!
• Antiporters (a.k.a. exchangers)!
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Membrane transport proteins
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Passive: only move through passive energetics
Ion channels: let charged things through open using aqueous pore
Diffusion channels: Let only uncharged things like glucose through
Active: ATP: Move things through a concentration gradient or electrochemical gradient/potential (from a lo…
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Channels vs. Carriers
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Channels function by forming a pore through the membrane
When channel gate is open many things can flow through at a extremely high rate
Carrier Proteins don't have open gates, they have a cyclical gate that works one at a time to selectively let compounds through at a slower rate t…
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Kinetics of Transport (11-7)!
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There is a maximum rate at which transport proteins can let things through, it's their saturation point "saturibility", this is because there are a limited number of proteins on a membrane and eventually they reach their maximum solvent movement rate
however diffusion doesn't peak or …
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Cells maintain an internal ionic environment that is different than the external environment!
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Notice that none is the same inside and out!
Total cations and anions are equal- electroneutrality!
Total number of particles inside and out is equal- osmolarity!
is the same!
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The importance of maintaining ICF osmolarity equal to that of the ECF
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ICF is intracellular fluid, ECF is extracellular fluid
ECF is very tightly maintained to maintain osmolarity to maintain healthiness of the cell
If cell is hypertonic to the solution then the water will flow into the cell -->swell/lyse
If cell is hypotonic to water, then water will flo…
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Ions and electrochemical gradients !!
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• For uncharged molecules like glucose, transport is simply based upon concentration gradient!
• For ions, charge adds another force!
– If the inside of the cell has a net negative charge, a negative ion (anion) will be repelled and a positive ion (cation) will be attracted!
– If t…
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Electrochemical Gradients (11-4)!
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Thickness of green arrow shows magnitude of the EC gradient, more or less traveling ions to within the cell
Positive potential inside counteracts the net traveling ions through the membrane, thus less ions flow through
There is an elec. potential across the plasma membrane (usually neg …
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Diffusion of ions gives rise to electrical potentials!
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• As K+ ions flow out, Cl- ions that helped maintain charge balance are left behind.
• These Cl- ions distribute themselves beneath membrane, attracted to cations outside. They exert a force across membrane. That force=electrical potential- membrane potential (Vm).
• When density of …
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MCB 2210: EXAM 1