Cell Structure Exam 1 Study Guide

Clathrin-coated vesicles

Endo and exocytosis 1) Arf1 GTPase 2) Cargo attach to Receptor → Adaptor protein attaches to receptor (ADP 1 → golgi to membrane/ ADP2 for retrieval or recycle) 3) Clathrin coat binds to adaptor protein 4) Dynamin 5) Vesicle formation Arf1 → Adaptor → Clathrin → Dynamin

Definition of Epigenetics

Epigenetics = the study of heritable changes in gene expression required that does not depend upon a DNA sequence

How does membrane fusion occur in the endomembrane system?

"Pierce and pull" 1) Tethering, docking, fusion 2) SNARE proteins pin membranes close together AND help *fuse* by promoting changes that allow transition past a hemifusion intermediate

What is an agonist, antagonist, inverse agonist, and biased agonist? How is the cellular response of cells will be when stimulated by each of them, in comparing to physiological ligand-GPCR interaction?

*Agonist* = enhances cellular activity *Antagonist* = competes for binding site of receptor and block cellular activity (*no response*) *Inverse agonist* = binds to same receptor as agonist but has *opposite response* *Biased agonist* = selective activation of a subset of the receptors signaling outputs

What is an amphipathic helix? Why are they present in a number of proteins involved in vesicular trafficking?

*Amphipathic helixes* = bend membranes, sar-1 provides mark to show where they bend Amphipathic helix allows insertion of protein into the lipid bilayer (causes curving of membrane, Sar1/Arf1 does this) *BAR proteins* = Bin, Amphiphysin, RVS Sensitive to membrane curvature, have higher affinity for more curved regions of the membrane, some have been shown to induce membrane curvature

GPCR signaling desensitization

*Arrestin* contributes to the desensitization process by: 1) prevents the activated receptors from interacting with g proteins 2) serves as an adaptor protein to help couple the receptor to the clathrin dependent endocytosis machinery

Be able to compare and contrast heterochromatin versus euchromatin, their relative effects on gene expression and nuclear localization

*Euchromatin*: 1) HIGH histone acetylation 2) LOW DNA methylation 3) H3-K4 methylation 4) Nuclear core *Heterochromatin*: 1) LOW histone acetylation 2) DENSE DNA methylation 3) H3-K9 methylation 4) Nuclear periphery

What is a GTPase? How is it regulated in terms of activation and inactivation? (3 types of important regulatory molecules) Which protein in GPCR system is a GTPase?

*GTPase*= protein that acts as a molecular switch; has an intrinsic rate of hydrolysis, intrinsically off *GAP* stimulate GTP hydrolysis (decrease on state = OFF) *GEF* catalyze the dissociation of GDP and exchange for GTP (increase on state = ON) *GDIs* inhibit the release of bound GDP (increase OFF state = OFF) GTP = ON, GDP = OFF

What is a kinase? Phosphatase? How do these two types of enzymes work to modulate signaling protein functions?

*Kinase* = covalently adds one or more phosphate groups to a specific AA on the signaling protein *Phosphatase* = removes phosphate groups Kinase = ON Phosphatase = OFF Work together to start kinase cascade (see next question)

How are cargos chosen for lysosomal sorting (e.g., signals)? What route do they take to get there?

*M6P = signal for TGN → lysosomal/endosomal sorting* Mannose-6-phosphate (M-6-P) signals the Trans-Golgi Network for lysosomal/endosomal sorting Protein (e.g. lysosomal hydrolase) has a terminal mannose residue on its N-linked oligosaccharide --> GlcNAc phosphotransferase (in the ER) adds GlcNAc-P to the to the mannose residue--> Protein is then transported to Golgi --> Golgi removes GlcNAc --> Remaining M6P tag on protein binds to M6P transmembrane receptor in Gogi --> signals for lysosomal/endosomal sorting

Understand nuclear import cycle (NLS, carriers, release). What type of assays were developed to elucidate the process.

*NLS* = sorting signal that prescribes delivery into the nuclear compartment - Fluorescent tagged protein NLS No cytoplasm = no nuclear transport Cytoplasmic extract = nuclear transport Each cargo has specific NLS →beta karyopherins or NLS → adaptor → beta keropherins Beta keropherins recognize NPC & bind FG repeats; have affinity for the nucleus NLS + Cargo protein binds to → import receptor → Ran GTP- cargo protein/NLS →receptor g TP complex → -ran GTP

What is a SH2, PTB, SH3, PH domain? Binding to what motif? What about C2 domain?

*PTB*= phosphotyrosine binding *SH2*= Sc homology 2 = phosphate binding = part of Grb2 adaptor protein *SH3* = Sc homology 3 = short proline-rich sequences = part of Grb2 adaptor protein *PH* = Pleckstrin Homology = recognize PIP3

What is the Rayleigh criterion? According to the criterion how does the wavelength of light affect the ultimate resolution?

*Rayleigh criterion* = determines the lower limit of resolution of the light microscope; wavelength limits your resolution; resolution is proportional to wavelength For two points to be considered distinct, the max of one must be further than the first dark interference ring of the other

What is a sec mutant? What is involved in doing an epistasis experiment? What information do such experiments provide?

*Sec 1 mutant* results in accumulation of secretory vesicles in cytoplasm E.g sec1 cells accumulate vesicles, sec 22 have engorged ER, what do sec1/sec22 double mutants look like If double mutant looks like sec1 --> accumulation of vesicles occurs first If double mutant looks like sec22 --> engorged ER occurs first (They look like sec 22 --> sec22 function precedes sec1 function) *Provides the relative order of these molecules in the secretory pathway*

Important features of 2nd messengers (familiar with names of well-known molecules?) and how do these features contribute to functions of 2nd messengers? Are all second messengers have to be water soluble?

*Second messengers* = small (= diffuse easily) chemicals generated in large amounts in response to receptor activation and diffuse away from their source, spreading to other parts of the cell; have low affinity for their targets e.g. cAMP, cGMP, IP3, DAG, Ca2+ cAMP and Ca2+ = water soluble and diffuse in cytosol diacylglycerol = lipid soluble and diffuse in plasma membrane

What are two means by which out of focus light is removed from images from thick samples? How do these microscopes or processing techniques work?

1) *Deconvolution* = computational removal of out of focus light based on the behavior of light. Uses a PSF and algorithm to deconvolve the image data such that it appears as a 0.1 micron bead 2) *Confocal microscope* = use "confocal" pinholes in microscope to focus on specific area

What processes affect net plasma membrane growth and or shrinkage?

1) *Exocytic pathway* = surface membrane *growth*; delivers secretory vesicles containing lipids, receptors, ion channels & other membrane proteins to the plasma membrane; also carries soluble cargos in vesicle lumens for release into extracellular space 2) *Endocytic pathway* = surface membrane retrieval (*shrinkage*); uptake of macromolecules required for biosynthesis

What is FRAP and FRET? What can they be used for? Freeze fracture EM?

1) *FRAP* = check whether lipids or proteins are static or move around; rate and how fast molecules are recovered - Pick an area and bleach 2) *FRET* = Foster Resonant Energy Transfer - Measures protein-protein interactions and *membrane fusion events*; very dependent upon proximity of donor and acceptor - Before fusion green donor emission is quenched by acceptor, after fusion = dilution of acceptor occurs and green donor emission increases - Membrane fusion is reported as a loss of FRET as a function of time In the *freeze fracturing* process, a specimen is frozen rapidly and cracked on a plane through the tissue. This fracture occurs along weak portions of the tissue such as membranes or surfaces of organelles. After cleaving, both surfaces are shadowed with a platinum film. This coating produces a replica of the surfaces.

Be able to describe the basic subnuclear structures and their derivation where applicable (e.g., Nucleolus, nuclear lamina, nuclear pore)

1) *Nucleolus* → Site of ribosome subunit production (rRNA) - Fibrillary centre → 45s pre-rRNA is translated - Dense fibrillary component → 45s is spliced → 5.8,18,28 subunits - Granular component → pre-rRNA maturation → pseudouridylation, ribo-methylation 2) *Nuclear lamina* → provides structure to nuclear membrane/pores - Composed of dimers/tetramers - Lipidated → attach to inner leaflet of nuclear membrane → tether heterochromatic chromosomes 3) *Nuclear pore* → nuclear import/export - Nucleoporins → Hydrophobic → sea of FG (phenylalanine/glycine) repeats → involved in brownian movement of b-karyopherins+cargo - Allow <5K dalton molecules to diffuse freely - Involved in b-karyopherin mediated import/export

What is a proto-oncogene, oncogene, tumor suppressor? Can you give several examples of proto-oncogene/tumor suppressor that were mentioned in the lectures (III)

1) *Proto-oncogene* turns into an oncogene - Proto-oncogene → mutation → oncogene Not every gene has the potential to turn into an oncogene 2) *Oncogene* = gene that promotes tumor growth, usually a mutated cell proliferation/growth gene - Mutated Ras→ removes GAP ability on Ras →Ras is always active (ON) 3) *Tumor suppressor gene* = a gene that inhibits/prevents tumor formation - Usually promotes apoptosis Ex: PTEN, P53, Tsc2

How does intracellular Ca++ level been kept low in general?

1) *Quiescent cells* - Plasma membrane associated Na+/Ca2+ antiporter and an active P class Ca2+ pump move Ca2+ outside the cell 2) *Cells that are recovering from a recent spike in signaling induced (Ca2+) increase, several mechanisms function to return the cytosol levels to basal* - ER has active pump that clears cytosolic Ca2+ - Mitochondria have H+/Ca++ symporter Many cellular proteins chelate (bind free Ca2+)

What molecules/signals are involved in vesicular targeting? Why are combinations of these signals required for proper targeting?

1) *Rab-GTP (Rab-GEF)* - Vesicle Rab → helps dock SNARES - Membrane target Rab → PI3K activation → Formation of competent target raft 2) *v/t-SNARES* → Membrane proteins that provide for both catalysis and specificity of the fusion reaction. - Come as cognant pairs: v-SNARES (vesicular); t-SNARES (target) 3) *Phosphoinositols* → helps recruit Rab proteins - fusion competent membrane domain assembly

What are the competing models for movement of cargo through the golgi apparatus?

1) *Vesicular transport model* - Golgi stacks are static and long-lived - Movement of cargo is mediated by transport vesicles --> *anterograde* COP I moves cargo forward and resident protein backward 2) *Cisternal Maturation model* (supported in yeast) - Golgi stacks are dynamic - Continual additional of material at cis-face --> creates new cisternae - Continue loss of material at trans face --> releases old cisternae - *Retrograde* COP I traffic moves resident Golgi protein to appropriate compartment - Cargo is moved along with bulk flow of the stack Visualization of Golgi maturation suggests that *cisternal maturation* occurs

Sphingolipids

1) 3-carbon backbone but one tail is part of sphingosine 2) the other FA is linked by an amide bond 3) the head is ether linked C1 has the head group, instead of C3 like in glycerophospholipids

Gαiβγ—Cardiac muscle relaxation

1) Acetylcholine binds to Muscarinic acetylcholine receptor (mAchR) on cardiac muscle cell 2) Gαi catalyzes exchange of GDP for GTP and *decrease in cAMP* 3) βγ dimer is activated and diffuses to K* channel 4) Association of dimer with K+ channel allows K+ efflux resulting in hyperpolarization of cell 5) Hyperpolarization reduces muscle contraction

Nitric Oxide Pathway

1) Acetylcholine is released from ANS 2) Leads to activation of cytosolic signaling protein Nitric Oxide Synthase (eNOS) 3) NO ligand then diffuses to target muscle cells and binds receptor/signaling protein *guanylyl cyclase* 4) Guanylyl cyclase activity produces cyclic GMP (cGMP) which serves as second messenger RELAXES

Lipid Kinase (PI3 Kinase) Signaling Pathways

1) Activated TRK activates PI3 kinase 2) PI3 Kinase catalyzes the phosphorylation of PIP2 on the 3rd position --> PI(3,4,5)P3 3) PIP3 serves as docking sites for signaling molecules, assists in the assembly of signaling complexes downstream of both GPCRs and RTKs 4) PIP3 recognized by PH domains (PDK1 and Akt) 5) Akt is activated and dissociates 6) Akt promotes survival by phosphorylating a class of proteins including *Bad* (inhibits it)

Coated Vesicles

1) COP II 2) COP I 3) Clathrin

How is vesicular cargo (membrane and luminal) selected for inclusion in particular vesicles? What types of signals are necessary on the cargos?

1) Cargo selection = *adaptors* filtering and selecting cargo and serving as an interface for coat assembly 2) Coat formation, fission and coat dissolution = *scaffold coat proteins* that bind and sculpt the ER membrane into a bud

What are the types of intercellular signaling modalities? (lecture I, slide 23). How in each case, does ligand concentration reaching to the target cells associated with ligand-receptor binding affinity, signaling reversibility, and types of cellular response?

1) Contact dependent= requires cells to be touching 2) Paracrine = depends on local mediators that are released into the extracellular space and act on neighboring cells 3)Synaptic = performed by neurons that transmit signals electrically along their axons and release neurotransmitters at synapses 4) Endocrine = depends on endocrine cells which secrete hormones into the bloodstream for distribution throughout the body Types: - Juxtacrine = cells in immediate contact - Autocrine = signals to self - Paracrine = short to mid range - Endocrine = long range based on diffusible ligand in blood

Epinephrine-Induced GPCR

1) Epinephrine (adrenaline) signaling results in mobilization of glycogen --> glucose which is released into bloodstream for use at distal sites 2) Sharp response is conferred not only by the sharp cAMP dependent response of PKA (allosteric cooperatively) but also by virtue of the coordinated shutdown of glycogen biosynthesis coupled to the activation of glycogen breakdown 3) two states: - *increase cAMP* = PKA active - *decrease cAMP* = phosphatase active glycogen phosphorylasekinase = increases glucose synthesis glycogen phosphorylase= increases glucose synthesis inhibitor of phosphoprotein phosphatase = increases glucose synthesis AKAPs localizes cAMP PDE and PKA to ensure PKA is inactive keeping cAMP levels low

Insulin-induced RTK

1) Involved in glucose uptake and glycogen synthesis 2) Activation of the insulin receptor results in Akt-mediated phosphorylation and inactivation of GSK3 (glycogen synthase kinase) - Phosphorylation of GSK3 is inhibitory = net increase in glycogen synthase activity = glycogen synthesis 3) Akt phosphorylates RabGAP localized to recycling endosomes

RTK-mediated signaling pathways

1) Ligand-receptor binding: - Ligands are dimeric which highlights how these receptors transduce a signal across the cell membrane - Binding of ligand does NOT directly lead to association with an intracellular signaling molecule, but to another *receptor unit* 2) Activation (trans-autophosphorylation) - Once the dimeric ligand is bound, *dimerization* brings the kinase domains close to phosphorylate each other - Trans-phosphorylation induces conformational changes for full activation of the kinase domain 3) Cross-phosphorylation - Fully activated RTK domains then phosphorylate one or more tyrosine sites on *other receptors* - creating docking sites for signaling

Know the range of resolution provided for by the various forms of microscopy - light microscopy, super resolution microscopy, electron microscope

1) Light microscopy = *0.2um - 0.5 um* (500 nm) 2) Super resolution microscopy = *0.02um - 0.05um* (50 nm) 3) Electron microscope = *0.001um* (1 nm) 4) Atomic microscope = *2nm*

Golgi Apparatus

1) Major processing station of oligosaccharides 2) Site of O-linked Glycosylation and Proteoglycan synthesis 3) Major sorting station for vesicular traffic in cell both outbound and inbound (cis and trans network)

General idea of which major signaling pathways are for cell growth/proliferation, which is (are) for survival, which is (are) for differentiation?

1) Map Kinase for cell growth 2) RTK pathways are typically for growth/proliferation as well as for cell survival PI(3)K/Akt = Cell survival and cell growth

Be familiar with the DNA base modification 5-methyl Cytosine, how it is generated, what its general effect is on gene expression, and how such an epigenetic mark is removed

1) Methylation of Cytosine occurs at Cytosine-Guanine pairs (CpG) Generated via DNA Methytransferase (DNMT) → Recruits Histone Deacetylase (HDACs) → removes acetyl groups on histone → chromatin condensation → Heterochromatin (unexpressed) 2) removed by demethylases of the *Tet* gene

What are the examples for intracellular receptor-mediated signaling? Why do they have intracellular receptors? How do they work differently from membrane-bound receptor mediated signaling? Why? What is in common?

1) Nitric Oxide Pathway (short term) 2) Steroid Hormone Pathway (long term)

How is receptor mediated endocytosis involved in regulation of signal transduction pathways?

1) Receptor Sequestration - Control of receptor or channel availability 2) Receptor Down-Regulation - Receptor removal and destruction

What kinds of forces mediates interactions between ligand and receptor, as well as most protein-protein interactions? Why is this concept important for understanding signaling?

1) Receptor ligand complexes involved multiple weak non-covalent interactions and molecular complementary 2) Forces involved = ionic, VDW, hydrophobic interactions 3) Important for ensuring specificity through maximization of weak interactions, interactions are dynamic not fixed

Mechanisms for signaling desensitization: principles and examples (both in GPCR signaling, RTK signaling, and in JAKS-STAT signaling)

1) Receptor sequestration 2) Receptor down regulation 3) Receptor inactivation 4) Inactivation of signaling pathway 5) Production of inhibitory protein

Lysosomes

1) Responsible for degradation of discarded cellular macromolecules 2) Substrates for lysosomal digestion derives from 3 major sources - Endosomes - Autophagosomes - Phagosomes

Gαt-transducin—Rhodopsin-mediated photon recognition

1) Rhodopsin (the GPCR) absorbs photon and is activated 2) Gαt-transducin catalyzes exchange of GDP for GTP (now active) 3) Active transducer dissociates and activates cGMP-Phosphodiesterase 4) Phosphodiesterase closes the cGMP-dependent channels and and results in membrane hyperpolarization

What are the common polar head groups on glycerophospholipids?

1) Serine 2) Ethanolamine 3) Choline 4) Glycerol 5) Inositol

Understand the basis of nuclear important including how differently sized cargos would behave (i.e., what size cargos are gated, which diffuse freely with no gating)

1) Small molecules = diffuse freely through the NPC (*less than 5kD*) 2) Large molecules = undergo an energy dependent translocation, diffuse much slower (*larger than 40-60kD*); requires GTP

Glycerophospholipid

A lipid containing a glycerol backbone with two fatty-acid "tails" and a phosphate-linked "head"

How does diet and environmental agents affect the progeny they have and how is this reflected in the epigenetic status of the CpG islands in these progeny?

BPA (*environmental*) → Inhibit methyltransferase activity → No CpG methylation → Expression of agouti gene → yellow D12, Choline, Folic acid (*diet*) → encourage methyltransferase activity → CpG methylation → Inhibition of agouti gene → not yellow

Why is the notion that the ER lumen is topologically equivalent to the extracellular environment correct?

Because during fusion/fission of a vesicle with the plasma membrane *preserves the orientation of the integral proteins* embedded in the vesicle bilayer

COP I Vesicles

COPI (Golgi → ER) 1) Cargo proteins containing amino acid sequence KDEL → bind to KDEL receptors 2) KDEL receptors have KKXX sequence that allows the binding of COPI proteins → vesicle formation 3) Arf1 is the GTPase

COP II Vesicles

COPII *(ER → Golgi)* 1) Sar-GEF on ER membrane activates cytosolic inactive Sar-GDP [GDP -> GTP] 2) Activated Sar-GTP inserts helix into membrane 3) Sar-GTP recruits Sec 23 4) Sec 23 recruits Sec 24 → which binds to cargo receptor 5) Sec 13/31 binds to Sec 23/24 → Produces coat formation → Vesicle formation 6) Hydrolysis of Sar-GTP [GTP-->GDP] releases coat proteins

What molecules are involved in receptor mediated endocytosis?

Can involve either clathrin or caveolin, uses AP2 adaptor & clathrin pits cover about 2% of total plasma membrane area

Why are conditional mutations required for the isolation and study of genes affecting events associated with vesicular trafficking?

Conditional mutations = active and inactive at different temperatures; can go back and forth between the two Has wild-type (or less severe) phenotype under certain "permissive" environmental conditions and a mutant phenotype under certain "restrictive" conditions.

What is the importance of contrast in microscopy? Why is it so important in looking at cells?

Contrast allows you to easily differentiate different components of the cell (e.g organelles)

Be familiar with the components of chromatin as described in lecture

DNA --[histone]--> nucleosome --[mutiple nucleosome]--> chromatin Histone protein → 4 subunits → H2A,H2B,H3, H4

Be aware of the possible effects of epigenetic modifications on the condensation of chromatin, and how condensation, when occurring near promoter regions near genes can affect transcription and thus expression of those genes.

DNA methylation --> condensed Histone deacetylation --> condensed Histone methylation --> condensed Condensed chromosomes near promoter regions inhibit gene expression

What is a lysosomal storage disease? What kinds of molecular defects give rise to these diseases?

Disease characterized by an accumulation of lysosomal substrates in lysosomes; specific lysosome enzyme doesn't make it to the lysosome substrate 1) *Trafficking mutation* - E.g I-Cell disease --> enzyme without recognition marker --> enzyme release to plasma membrane --> no recapture 2) *Substrate Processing enzymes* - E.g Hurler syndrome --> No enzyme production Problem: Given what you know about lysosomal trafficking how is it that when you co-culture WT and I-cells or Hurler cells they can overcome their lysosomal storage phenotype? [enzyme replacement therapy] Providing one or more lysosomal enzymes in extracellular medium can correct deficiencies in mutant cells

What are the different ways membranes can be bent? What does this bending achieve?

Five ways: 1) Lipid composition 2) Membrane proteins 3) Cytoskeleton 4) Scaffolding 5) Helix insertion Curved or bent membranes are the most *dynamic* (for budding)

What is fluorescent protein technology? What does it permit that normal immunofluorescent microscopy does not?

Fluorescent fusion protein (in contrast to immunofluorescence) does NOT use antibodies to "tag" proteins. Rather, the DNA sequence for the production of a fluorescent protein (e.g GFP) is added next to the DNA sequence of the desired protein (e.g myosin) → so when the myosin gene is transcribed and translated so is the GFP gene. This results in a myosin protein FUSED with the GFP molecule allowing the protein to fluoresce.

What is the principle of fluorescence?

Fluorescent molecule absorbs a specific wavelength light (blue) → emits *longer wavelength* light (green) of lower energy. This allows you to observe fluorescently tagged objects. E.g GFP tagged receptor → When you illuminate entire sample with blue light ONLY the GFP tagged receptor will give a signal → green light

Phosphotyrosine (pY) residues

Form docking sites for downstream intracellular signaling proteins The process of cross-phosphorylation yields an array of sites which form the basis for complete assembly of receptor-mediated signaling proteins Thus, activated RTKs possess an intrinsic *activation-specific scaffolding* function In turn, these proteins may themselves be phosphorylated (increased activity) and serve as binding sites for other signaling proteins Ultimately, a signaling complex is assembled

Where in the genome is 5-methylCytosine found both with respect to genes and on a more global level within the nuclear compartment? What types of processes is it involved in controlling?

Found in heterochromatic regions like centromere and telomeres as well as repressed genes along the chromosome arms Processes: 1) Control of gene expression 2) Chromosomal stability 3) Cell differentiation 4) Genetic imprinting 5) X-inactivation 6) Carcinogenesis 7) Aging

what is the role of each subunit of the G protein? what are the types of G protein?

G-alpha = GDP/GTP G-alpha and G-gamma = heterodimer functions as a GDI (guanine dissociation inhibitor) keeping alpha subunit in (off state) Both G-alpha(ATP) and beta/gamma dimer function to *relay signals* when activated

Understand GTPase cycles, the effect of accessory factors

GAP stimulate GTP hydrolysis (decrease on state = OFF) GEF catalyze the dissociation of GDP and exchange for GTP (increase on state = ON) GDIs inhibit the release of bound GDP (increase OFF state = OFF)

Relationship between receptor and G protein, coupled or not? What coupled G protein might contribute to the signaling? What is the contribution of receptor to G protein function?

GPCR is coupled to a heterotrimeric G protein (composed of G-alpha, G-beta, G-gamma)

Understand there are two types of strategies to manipulate the activity of a signaling protein

Generate mutant to become either *constitutively active* or constitutive inactive (*dominant negative*)

Pathways regulating activity of Phospholipase Cβ - Gαq :Ca++, Protein Kinase C (PKC)

Gq protein stimulates Phospholipase C Phospholipase C cleaves PIP2 into IP3 and DAG IP3 binds to lumen of ER and causes release of Ca2+ Ca2+ and DAG bind and activate Protein Kinase C

Pathways via cAMPs as 2nd messenger - Gαs Gαi

Gs proteins stimulate adenylyl cyclase (ATP --> cAMP) Gi proteins inhibit the adenylyl cyclase cAMP binds to PKA and activates

Triacylglycerols

Have 3 fatty-acid tails bound to a glycerol backbone and no head (makes it very hydrophobic) 1) most abundant class of lipids 2) not a component of membranes 3) fats and oils in plants & animals 4) energy reservoirs in animals 5) fatty-acid triesters of glycerol Triglycerides with one type of fatty acid chain are simple; while those with different chains are mixed

What does development of a cell free system (aka in vitro reconstitution) for a particular biological process allow researchers to accomplish?

In vitro assays were created in order to elucidate a distinct biological mechanism for the process of vesicle budding transport E.g *ER vesicular formation* 1) Isolated ER membranes (differential centrifugation) --> Incubate membranes with fractions/protein + NTPs --> yields vesicles --> vesicles isolated by centrifuge 2) See which fraction/protein yields what kind of vesicles

How is Kd defined and what does it represent for in terms of ligand-receptor affinity? How to interpret results from the radioligand binding assay? What are the relationships among the amount of ligand-bound receptor, Kd, and cellular response? Why?

Kd= ½ of ligand bound to receptor Lower Kd = higher affinity Kd= equilibrium dissociation constant Radioligand binding assay: 1) Curve A = maximal # of *radioactive ligand*. All receptors are occupied by radioactive ligand 2) Curve C = measure of same concentration of radioactive ligand PLUS increasing concentrations of *unlabeled ligand* 3) Curve B = generated after substring Curve A - Curve C, to measure specific binding and associated Kd

What steps of signaling amplify the initial signal?

Kinase cascades = phosphorylation → kinase 1 → phosphorylation → kinase 2 and so on Amplification of the signal Intracellular signaling molecules are usually involved in amplification The more steps in the signal transduction cascade = more amplification

Why does something like a 1 nm diameter fluorescent molecule appear to be at least 200 nm in diameter in a light microscope?

Limit of resolution for light microscope; *diffraction limited resolution*

Why are misfolded proteins inconsistent with cell function? What types of molecules monitor and catalyze protein folding?

Protein function is HIGHLY LINKED with protein structure Misfolded protein → incorrect protein structure → lack/incorrect protein function *Proteins must be properly folded in order to transit to the Golgi* *Chaperones (e.g BiP & calnexin)* → bind to the exposed hydrophobic region and unstructured peptide backbones (usually shouldn't be exposed in properly folded proteins) of misfolded proteins → Use ATP to attempt to refold → may take many cycles Quality control of misfolded proteins occurs in the ER --> *ERAD* (ER assisted degradation) by *retrotranslocation*

Epigenetic changes alter the expression of genes. Many epigenetic modifications occur in the context of chromatin. Be aware of the types of modifications but don't been deeply concerned with the details of the "meaning" of these modifications to the transcriptional machinery. Also, do not concern yourself with knowing the histone code, that is, where on the histone tails these modifications occur.

Methylation, Acetylation, Phosphorylation *Intrinsic effects*: Acetylation of Lysine (K) and Methylation of Arginine (R) changes the effective charge of these basic Amino Acid residues --> Interferes with nucleosome-nucleosome interactions (condensation) *Extrinsic effects*: Modifications present on the histone tails also serve as binding sites for enzymatic activities that further alter the availability of the DNA to regulatory proteins

Phosphatidyl Serine

Negatively charged (-1) 1) Important for the binding of proteins to membranes 2) Found in the inner leaflet of the plasma membrane

Phosphatidyl inositol

Negatively charged (-1) (important for the binding of some proteins to membranes) 1) the OH hydroxyls on the 3, 4, and 5 position of the inositol ring can be phosphorylated. 2) Inositol lipids are very important in *signal transduction*

Be familiar with signaling pathway components regulating glycogen-glucose metabolism under different circumstances (Epinephrine-induced GPCR vs. insulin-induced RTK)

Next question(s)

How does PALM microscopy work? Why is Photoactivatable GFP used in the examples we discussed

PALM = photo activation localization microscopy 1) Image then bleach subpopulations & repeat 2) Image constructed by summing position probability gaussians determined for all data PALM microscopy = fluorescence microscopy imaging methods that allow obtaining images with a resolution beyond the diffraction limit Photoactivatable GFP is much brighter than regular GFP (100x) (absorb longer wavelength) → May also aid in only bleaching a subset of molecules

What is a point spread function?

PSF = represents how the light behaves in a microscope and how the light is convoluted out of the plane of focus, used to *deconvolve* the image and remove out of focus information

Describe the structure of the nuclear pore and the relevance of Nucleoporins.

Nuclear pore → nuclear import/export *Nucleoporins* → Hydrophobic → sea of FG (phenylalanine/glycine) repeats → involved in brownian movement of b-karyopherins+cargo - Allow <5K dalton molecules to diffuse freely - Involved in b-karyopherin mediated import/export - Larger molecules undergo an energy dependent translocation (around 40-50kD or greater) - 8 fold symmetry

What do stains accomplish?

Provides contrast; rather than all of the specimen being translucent, now different parts absorb/reflect different amounts of light.

What types of molecules would you expect to find in fusion competent membrane domains?

Rab-dependent formation of fusion competent domain results in the presence of all 3 components of the code (see above question)

The role of Ran asymmetry in concerted transport

Ran asymmetry represents the "gradient" of GAPs outside (Cytoplasmic) and GEFs inside (nuclear), forcing transport to occur in one way

What is Ras? How does it work in signaling? How was it discovered?

Ras = important signaling protein that can activate various downstream signaling pathways (*Small GTPase*) Functions as a molecular switch, cycling between the two distinct conformational states Active = GTP bound, inactive = GDP bound Ras = proto-oncogene ??

Why are retrieval pathways required in vesicular trafficking pathways? What do they retrieve?

Retrieval pathways exist to recycle both the lipids and non-cargo elements in the biosynthetic secretory pathways Retrieved *Cargo receptors* must be brought back to the ER if they are to be reused Retrieval is specified by specific signals (e.g. *KDEL receptor*)

Pathways regulating activity of Ion channels: (1) Gαiβγ—Cardiac muscle relaxation (2) Gαt-transducin—Rhodopsin-mediated photon recognition)

See next question(s)

Why are mutations that affect cell division in yeast in genes that are involved in vesicular trafficking?

Similarities: 1) Haploid eukaryotes 2) Fast generation time 3) Easily mutagenized 4) Very much like us even though we are separated by 109 years 5) Have all the same basic parts that we have: golgi, ER, nuclear membrane, double bilayer *Cell division requires membrane fission → same mechanism used for vesicular fission*

Most of signaling effectors get combinatorial codes (multi-phosphorylation, for instance), what does this contribute to? Specificity or reversibility?

Specificity

Know the general structure of the Agouti viable yellow allele. What relevance do CpG islands have to the expression of the yellow gene in this mouse? What type of progeny do these mice give rise to (coat color). How is it that despite having identical DNA sequences some progeny are yellow and some are pseudoagouti?

Structure → Retrotransposon promoter adjacent to Agouti gene CpG islands near the Retrotransposon silence the gene when methylated → thus inhibiting the expression of agouti gene Lack of CpG methylation near the retrotransposon → no inhibition → expression of agouti gene via retrotransposon promoter *Yellow* → No CpG methylation, *Pseudoagouti* → CpG methylation

What is the gene gating hypothesis?

The idea that the DNA sequences themselves might be segregated in a specific fashion to compartmentalize function 1) Euchromatin = *inside* 2) Heterochromatin = *outside*

Describe the organizing principles for chromosomes in the eukaryotic nucleus and relationship between chromosomal organization and gene activity.

There is some order established by virtue of the presence of nuclear organizing regions (NOR) which corresponds to the site of ribosomal DNA present on chromosomes 1) *Euchromatin* = loosely packed = high gene activity (Nuclear core) 2) *Heterochromatin* = highly packed = low gene activity (Nuclear periphery)

What is the mechanism contributing to signaling specificity and meanwhile preventing cross-talk?

This is due to *scaffolding proteins* → promote assembly of signaling complexes as well as anchoring molecules to appropriate sites → Prevents cross talk

Transport out of the Golgi

Three major pathways: *Lysosomal, plasma membrane, secretory vesicle* emanating from Trans-Golgi Network (TGN) In the absence of specific signals directing cargo to lysosome, secretory vesicles or retrograde recycling (back to ER?), cargo is directed to the plasma membrane

Phosphatidyl Ethanolamine

Uncharged 1) Enriched in the cytoplasmic (inner) leaflet of the plasma membrane 2) small head group allows membranes to curve

Phosphatidyl Choline

Uncharged 1) Plays a structural role in membranes 2) Concentrated in the extracellular leaflet of the plasma membrane

How do immunofluorescence experiments work? Know how you might look at the distribution of two proteins in the same cell via immunofluorescence

Use the property of antibodies (immuno-) to label objects. Two proteins (e.g Actin / Myosin) → anti-actin-GFP antibody will ONLY bind to Actin while anti-myosin-Cy5 antibody will ONLY bind Myosin. Illuminate sample with blue light → activates GFP → location of actin. Illuminate sample with red light → activates Cy5 → location of Myosin. Throw the two images together to get the location of Actin and Myosin; Use of both primary and secondary antibodies → AMPLFICATION OF SIGNAL

How does influenza virus enter the cell? What cellular molecule does it resemble with respect to its function?

Viral envelope protein, Hemaglutinin (HA) binds to cell membrane → causes endocytosis → endosome formation containing virus → Low pH 5.5 produced in endosome → Conformational change of HA protein is triggered by low pH → exposure of hydrophobic fusogenic helix → Viral membrane fusion occurs → Virus delivered in the cell

Why do images from fluorescent microscopes yield fuzzy images from thick sections?

You are illuminating and transmitting light to the detector from the region *above & below* the plane of focus The sample is in 3D → so there is a y axis. When you illuminate the sample you are illuminating a COLUMN of fluorescent molecules → provides plenty of out of focus light → fuzziness

What kind of light microscopy is useful for looking at living cells?

You can't stain living things so: 1) Phase contrast microscopy 2) Differential interference contrast microscopy