MCB 252 Exam 3 - Exam 3 summary and lecture notes PDF

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Exam 3 summary and lecture notes...

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MCB 252 – Lecture 3/6  Properties of cells in tissues in order for the organ/tissue to function properly o Need a defined shape to carry out a certain function o Work together: cell adhesion: for one cell to stick to another cell, they need to adhere in some way. This is because cells need to be properly arranged. o Work together: coordinate activity and communicate o Remain separate from cells in neighboring tissues o Tissue homeostasis: our cells, tissues and organs need to be kept the right size and shape (to avoid damage or bursting of cells)  Keep tissues the same size and shape, and replacing dead cells with new cells o Carry out specialized functions (terminally differentiated cells)  Stem Cells o Stem cells divide and give rise to differentiated cells o Stem cells are undifferentiated cells. They go through the developmental process to create differentiated cells. o When stem cell divides, it gives rise to 2 cells. One of those is a stem cell and the other is a terminally differentiated cell (that can become another cell type in the cell)  Terminally differentiated cells can no longer divide. They are specialized for a certain function and they do that job for the rest of their life  Cells undergo several steps leading to terminal differentiation. They made need to divide many times before they become a specific cell type.  This is asymmetric cell division  Symmetric division is if the split cells are identical to each other.  The cell can go through either type of division  Ultimately, cells within a tissue need to communicate with each other. The cells need to divide at the correct rate to maintain the tissue as a whole.  Four global responses to signals (alternate fates of cells) o Survive, grow + divide, differentiate or die o Different signals cause these responses o Most signaling is from cells to each other o Cells needs signals to stay alive because it helps keep the tissues in the correct areas o Types of signaling molecules are growth factors, mitogens, and cytokines o If the cell adopts one fate (like divide), it blocks the others from happening  Apoptosis: programed cell death o It can happen in response to signaling in normal development  Required in many organisms (tadpoles use this to get rid of their tails)  This cell suicide is done by breaking up the DNA and recycling the cell’s parts so other cells can use them. o Apoptosis can also occur in response to cellular damage  If the cell is exposed to DNA damage, the cell cycle is stopped. If the cell can fix the damage, the cycle will be resumes and division continues. If the damage is too severe, cell suicide takes place.  When cells divide, cell adhesion to its neighbors doesn’t take place  The 4 processes are coordinated and inter-related o Tissues: adhesions, cell cycle = cell division, cell shape = cytoskeleton

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Properties of cancer o Inappropriate or uncontrolled cell growth causing tumor formation o Metastasis: break away from normal tissue (inappropriate adhesion) and move to other tissues (inappropriate movement) o Divide in new tissue to form secondary tumor  Here, they divide when surrounded by cells that are not normally their neighbors (inappropriate signal response) Control of cell division in development (comparing rate of cell division in 9 months of human gestation vs 9 months after birth) o An embryo grows at an exponentially faster rate than babies after birth o This shows that there are a wide range of rates of cell division and they need to be properly regulated during, before and after development Development in Xenopus Laevis (frog) o Cell cycle  The large fertilized egg is produced externally by the mother  Blastocyst: cell adhesion and movement is described in picture above o Movement of sheets of cells  Three primary tissues: ectoderm, mesoderm and endoderm  The sheet cells on the outside migrate around the dorsal lip and move into the egg  The dorsal lip is a signal receptor  The cells around the lip are signaled to differentiate  So, the ectoderm cells become mesoderm and endoderm cells  Cell-cell adhesion holds cells together to from sheets  The egg undergoes rapid rounds of cell division  The cells then continue to differentiation  The ectoderm sheet cells move and form into a tube that becomes the spine of the animal  The cells remaining on the outside will form structures like the skin

MCB 252 – Lecture 3/8  Many aspects of development can tissue formation can be viewed as converting sheets of cells to tubes, balls, or tubular solid structures o The 4 cell properties (cell mobility, adhesion, differentiation and division) control the development of cells  Continuing talking about cell shape o What doesn’t determine cell shape?  Bio-membranes don’t determine cell shape  example in lipids  Lipid bilayers in water form a spherical membrane that is fluid  These membranes can be easily deformed, they are tough to break and they are “self-healing”  This fluidity and easily deformed characteristic means the shape is not retained and this means the membrane can’t determine shape of the cell o What determines cell shape?  The cytoskeleton maintains and defines shape of cells by proving a rigid framework

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Cell motility is dependent on cell shape o Examples: sperm, pollen, cytokinesis, locomotion, phagocytosis and more o All these distinct types of movement are…  They are based on 2 types of filaments  Actin filaments and microtubules  They are based on 3 types of motors  Myosin, dynein, and kinesin o Cytoskeleton is comprised of 3 types of filaments, 2 of which are the same filaments that interact with motors Molecular evolution o Gene Families  Gene duplication  An accident like a chromosome breakage and rejoining causes something in the cell to go bad and a second copy of the gene is generated  The two separate copies pick up different mutations and become different genes.  This can happen multiple times during evolution  Gene divergence  Process by which genes are formed by going through independent genetic changes/mutations over time A gene family is created from a single gene that gets mutated throughout time to create various different genes  This can happen multiple times during evolution  Different family members have related functions but they can be specialized for their optimum protein function  The regulatory regions of genes can also evolve  The genes on the far left are most closely related and are arranged in clades by time to common ancestor  Family members have similar structure, function, and regulation o Protein domains  TFIIIA contains 9 zinc finger domains. Each domain is similar in structure but each finger recognizes different proteins.  Each zinc finger recognizes 3 bp, so the 9 fingers bind 27 nucleotides  Many proteins are constructed modularly by putting together different combinations of domains.  In general, the more complex an organism is the more complex the proteins are in terms of their protein architecture  One group of proteins are that particularly modular are eukaryotic TFs  Chromosomal accidents cause a region of one gene A to be inserted into a different gene B. After transcription and splicing, that region of gene A is a part of the gene B. So, the mRNA for the protein has a new domain.  This process does not occur in prokaryotes because the gene A would need to be inserted into the open reading frame, since prokaryotes don’t splice.

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As organisms become more complex and have more complex modular gene structure, alternative splicing is an important aspect of this process. Alternative splicing allows for different genes to evolve by adding or not adding certain genes. So, new domains can be created and old one can be retained.  Splicing allows exons to work over a distance and allows modular proteins to be built o Eukaryotic gene structure  Structure of regulatory regions  Have regulatory promoter, core promoter, coding region and enhancers  Enhancer can turn the gene on in response to a different signal or condition. There can be many different enhancers on a region.  Eve expression is drosophila o Eve transcription in different segments is activated by different enhancers. Txn in 7 segments is directed by 7 enhancers.  Regulatory regions also evolve in a modular fashion o They take place due to chromosomal accidents like mutations  Enhancers can be located anywhere on a gene and they will still be able to control the gene, regardless of precise location. Enhancers also work in any orientation. The ability of enhancers to work over any distance allows a gene to accumulate multiple enhancers and complex regulatory regions. NEW LECTURE MATERIAL MCB 252 – Lecture 3/11  Biochemistry: purify the proteins involves and study their behavior in vitro. Recapitulate the process in a test tube  Genetics: isolate a mutant that is defective in the process  Cell biology watch the process in a microscope. Follow the dynamics of particular proteins in a living cell. Does inhibiting function of certain proteins in living cells block specific processes? o All three of these processes are interrelated  Biochemistry  protein purification o First step is cell extraction  Place some tissue or cells in a buffer. Then, break the cells open so the proteins inside are released into the buffer.  This gives both soluble and insoluble material  Next is centrifugation to separate the soluble from insoluble material  All the insoluble material will precipitate at the bottom of the tube  The supernatant is the soluble material in the buffer (liquid at the top)  We will then take the supernatant and that is called a cell extract o Next step is Fractionation  Fractionation is separating proteins based on their physical or chemical properties  So, there are 1000’s of types of proteins and many copies of each type of protein in the cell extract

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This extract is separated based on chemical and physical properties  So, the proteins get separated and put into different test tubes  These test tubes go through further rounds of purification until the proteins come out purified. o This is done through column chromatography o Column Chromatography  It is a glass cylinder with a filter at the bottom. The column material is made up of small beads. The filter holds the bead in the cylinder and the beads make up the column.  The cell extract is added to the top of the column  Buffer is added and it is able to diffuse through the column. As more buffer is added, the protein separation takes place. The farther down the protein travels, the less is interacts with the column and the more rapidly it travels.  With increasing time and buffer, each protein will diffuse out separately and can be collected. Types of Standard Column Chromatography o Ion Exchange  Proteins are separated based on charge  Chemical groups are attached to the beads that make up the column. These groups can be positively or negatively charged.  Ex) If the beads are positively charged, the ions that are negatively charged will be attracted and will bind to the beads. The rest will pass through  To remove the proteins attached to the beads, a salt will need to be added in the buffer. Tightly bound proteins have a high charge and need a high salt concentration to be removed from the beads. o Gel filtration  Proteins are separated based on size  The beads have pores of different sizes so different sized proteins can be eluted from them  Large proteins elute fast because they cannot into the beads  Medium sized proteins will get through half of the beads and will take more time eluting. The pass around the beads slowly.  Small proteins fit through all beads and take the longest time o Elution Profile  The fraction number is the different test tubes on the bottom  The green line is the salt concentration and the red line is the protein that have been eluted (picture on the right)  The end (right part of graph) for ion is the high ion affinity proteins and for gel filtration, the end is the smallest proteins  In vitro assay: this biochemical test will help identify protein activity  Place the protein of interest and see if it gets broken down  Different proteins generate different biochemical assays. You will run the protein of interest in the assay and see which tubes generate a product. Multiple tubes will generate a pool and these tubes will be run over multiple columns until purified Example of how RNA Poly II was purified

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o Begin with a DNA (plasmid) + protein fraction + radioactive nts o If the fraction contains RNA Poly II, the radioactive nts will be put into a chain and this chain will precipitate in the presence of TCA o If the fraction doesn’t contain RNA Poly II, the radioactive nts won’t be put into a chain and there will be no precipitate in the presence of TCA  So, you are looking for the fraction in which radioactivity precipitates o It takes repeated rounds of purification in combination with an assay to end up with the purified protein of interest After purification, we will try to determine the structure of a protein o Structure provides insight into the function of the protein (two examples below) o Chaperonin: protein folding device  All information of protein folding is in the primary chain but many proteins need help being folded into their tertiary or quaternary structures  The chaperonin is a dome structure that opens to let the protein in and then closes. Inside the chamber, the misfolded protein is exposed to a chemical environment different from the cytoplasm. This allows the protein to re-fold. o DNA Helicase: separated DNA from ds to ss  Its donut shaped and the hole in the middle only fits ssDNA  This machine gets on dsDNA and uses ATP hydrolysis to move along the strand, pushing the two strands of DNA away from each other o How do we determine structure after purification?  X-ray crystallography (gives electron density map – not a picture of protein)  You take the purified protein, concentrate the solution, and see if crystals are formed. In a crystal, all the proteins are organized and stacked on top of each other in an orderly fashion  Next, you shoot an x-ray bean through the crystal  Due to this organization in the crystal, they will refract the beams in a particular way due to their shape  Then you do math with those refractions and determine the shape  You must know sequence of protein or DNA and fit it into the electron density map. And this will provide the structure How to determine localization of a protein within a cell o Immunofluorescence  One what you want to see is fluorescent, everything else is black  How to visualize one protein in a cell with thousands? o How to make antibodies 2: multiple antibodies are derived from a mouse cell o How to make antibodies 3: Monoclonal (unlike polyclonal, they are in high supplies in the cells).  Next, visualize the antibodies by attaching something to their constant region (this is the straight line, the tips are the variable region – epitope) o Radioactivity, Fluorescent “tag”, or Chemical reaction (enzyme)  The primary antibody (polyclonal) attached directly to the antigen. The secondary antibody (monoclonal) has the labeled tag. The secondary antibody binds to the primary  This fluorescence will be visualized with a fluorescence microscopy

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o It will be set to visualize only certain wavelengths, so certain colors o Different colors can be generated by repeating the experiment with different wavelengths of light o Visualize microtubules by immunofluorescence and DNA by a fluorescent dye (DAPI)  DAPI is blue when bound to DNA Some antibodies can bind and not inhibit protein function. Many antibodies can inhibit protein function. o Antibodies that block protein function are called “blocking” antibodies

MCB 252 – Lecture 3/13  3 techniques to put DNA into cells or organisms o Transfection = transient expression  Only works for a short period of time  Take the cloned gene and put it on a vector, which is inserted into a plasmid.  DNA remains on a plasmid and is maintained as an episome (non-chromosomal DNA) for a day or two and is then lost  So, if this circular DNA is maintained episomaly, the gene on the plasmid can be expressed for a couple of days only o Transformation = stable, long term expression  DNA is integrated into a chromosome and maintained indefinitely  Stable cell tissue culture lines can be created  The lines can be incorporated into genes so that DNA can be replicated, incorporated and segregated into that chromosome  It’s harder to make cell lines than incorporate DNA transiently  There is a low percentage of the DNA that gets in that can actually integrate. This process is more work in general. o Transgenic Animals  Integrate a gene into the genome in the gametes of an organism  All progeny in subsequent generations will contain that gene in all cells  Some uses of Transformation/Transfection/Transgenic o Put mutant forms of genes (and consequently proteins) into cells o Visualize cells and proteins via Green florescent protein (GFP) labeling and fluorescence microscopy  GFP provides a second visualization tool  GFP can be expressed in cells of other types and there is will fluoresce  One way to use GFP is to take a coding region of GFP and attach it to a specific promoter or tissues specific promoter. This will drive GFP transcription and allow the specific cell type (like flies or neurons) to be imaged in transgenic organisms  Another use of GFP is to make gene fusions, which make protein fusions. o So, the location of the gene within the cell can be traced by following the localization of the fused protein.  GFP allows imaging of live cells (can look at protein dynamics)  Immunofluorescence cannot be done on live cells o Antibody staining involves fixed (dead) cells

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There are many derivatives of GFP (different colors from mutational analysis)

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Electron microscopy defined 3 types of filaments in cells  Actin is a cytoskeleton filament (microfilaments) (smallest diameter)  Microtubules (largest diameter)  Intermediate filaments o They are all cytoskeletal polymers that assemble by protein-protein interaction (instead of by covalent interactions) o All the filaments are multi-stranded (actin has 2 strands, microtubules have 16, intermediate filaments have 13 strands)  Single stranded polymer is much shorter than multi-stranded Actin Cytoskeleton o Stable structures  Filaments have a long half-life and persist for a long time  Example is microvilli (you want it there for a long time)  Involved in cell shape and cell adhesion o Dynamic Structures  Filaments have a short half-life and are short lived  Lamellipodium (leading edge of the cell that is moving)  They are constantly being broken and re-made because its moving  Involved in cell movement Actin structure o It is a subunit of microfilaments o One of the most abundant intracellular proteins in eukaryotes o High conserved throughout evolution (very similar in distantly related organisms) o Different isoforms have different functions (humans have 6 different actin genes)  Alphas: muscle  Beta: leading edge of moving cells  Gamma: stress fiber o It is a 42 kD globular, ATP-binding protein (hydrolyzes ATP  ADP) o Monomer = G-actin o Apart from ATP & ADP, it can also bind ions like Mg2+, K+, or Na+  This binding changes the conformation of actin and causes the spontaneous polymerization to F-actin (polymerization is reversible)  Reversible assembly is critical for many cell movements  The 3 possible conformations of actin are bound with ATP, bound to ADP, or bound to no ion/nucleotide (like Mg) o Actin filament structure  Looks like twisted beads on a string (7-9 nm diameter)  Subunits arranged as a tightly wound helix  Can be described as short pitch – every subunit (coils)  Or as 2 long pitch helices (vertical line of subunits)  The distance equal to one full turn of an actin filament (long pitches) is 72 nm  36 nm is one half turn of the helix<...