Unit 2 Exam Flashcards
What are the main steps of the secretory pathway?
- Translates from mRNA in the ribosomes in the cytoplasm
- Enters the ER lumen
- Goes from the ER to the Golgi in a vesicle
- Transits the Golgi
- Leaves the Golgi in a vesicle
- The vesicle fuses the cell membrane
- It is outside
Give examples on how mutated proteins affect the secretory pathway?
- Fail ER import
- Fail to produce ER vesicles
- Vesicles don’t fuse to Golgi
- Fail to leave the golgi, Golgi vesicles do not form
- Vesicles don’t fuse with cell membrane
Describe the organization of the ER? Rough vs smooth?
organization
- netlike labyrinth of branching tubules and flattened sacs that extends throughout the cytosol
- ER has a single internal space, called the ER lumen
Rough ER
- has ribosomes bound to the membrane surface.
Smooth ER
- lack ribosomes
biosynthesis and metabolism of lipids.
What is cotranslational import?
Cotranslational translocation or import
- occurs when membrane-bound ribosomes insert growing nascent polypeptide chains directly into an ER translocation pore.
- targeting of cytoplasmic ribosomes translating signal sequence-containing polypeptides to the ER is mediated by the signal recognition particle (SRP).
Give an overview of protein secretion.
- Cotranslational import into the ER
- ER signal sequence is guided to the ER membrane by at least two components: a signal-recognition particle (SRP), binds to the signal sequence, and an SRP receptor in the ER membrane
- When a signal sequence binds, SRP exposes a binding site for an SRP receptor, which is a transmembrane protein complex in the rough ER membrane - Membrane-bound ER ribosomes make proteins that are co-translocated across the ER membrane.
- free ribosomes, unattached to any membrane, synthesize all other proteins - Polypeptide Chain Passes Through a Signal Sequence–gated Aqueous Channel in the Translocator (or Translocon)
- In multipass transmembrane proteins, the polypeptide chain passes back and forth repeatedly across the lipid bilayer - Translocated Polypeptide Chains Fold and Assemble in the Lumen of the Rough ER
What three things does Protein folding in the ER involve?
- Chaperone proteins
- Disulfide bonds
- post-translational modifications that occur in the ER - Glycosylation
- Addition of a Common N-Linked Oligosaccharide
- Oligosaccharides are used as Tags to mark the state of protein folding
Note on Glycosylation:
folded properly: remove last glucose
not folded properly: add a glucose
What is Calnexin? What does it do to incompletely folded proteins?
- It is a chaperon protein
- binds to monoglycosylated on incompletely folded proteins and retain them in the ER
- Recruits an oxidoreductase (ERp57) to add more disulfide bonds
- If folding is good, GlsII removes the final glucose residue
ER Chaperones prevent what? Benefits to this? Give examples of chaperones.
- Prevent protein misfolding and aggregation by giving misfolded proteins a second chance (to fold properly)
- Examples: Hsp70/BiP, Calnexin
Benefits:
- Protects peptides from interacting with other misfolded proteins
- Create a folding environment
How are misfolded proteins recognized?
- Misfolded proteins are recognized because they have exposed hydrophobic residues
- Hydrophobic residues should not be Outside
- Folding sensors recognize Hydrophobic surfaces
Explain what you know about transport vesicles?
- form from specialized, coated regions of membranes.
- bud off from one compartment, as coated vesicles (distinctive proteins in a cage), and fuse with another (carrying material as cargo)
- ER proteins have the KDEL sequence (Lysine - Aspartic acid - Glutamic acid - Leucine) and KDEL receptor initiates vesicle formation
Distinguish between where the secretory pathway and endocytic pathway lead?
- secretory pathway leads outward from the endoplasmic reticulum (ER) toward the Golgi apparatus and cell surface
- the endocytic pathway leads inward from the plasma membrane (replenishes vesicles)
Name the four well-characterized types of coated vesicles. Each type is used for different transport steps.
How do vesicles know they are loaded & good to go?
- Clathrin-coated
- COPI-coated
- bring ER proteins back from the Golgi - COPII-coated
- can accommodate large cargoes by assembling tubes instead of vesicles
- Proteins leave the ER in these vesicles as they form at the ER exit site - Retromer-coated.
Cargo receptors inside vesicles ensure they are loaded & can leave
Note:
- all transport vesicles display surface markers that identify them and target membranes display complementary receptors.
Explain what you know about the Golgi. What processes create destination codes?
Golgi
- consists of a collection of flattened, membrane-enclosed compartments called cisternae
Destination codes
- Created by glycosylation and phosphorylations
- Sugar coats serve as destination tags (some are transient)
- Glycosylation steps are
compartmentalized in different cisternae
By what two mechanisms does transport through the Golgi Apparatus occur?
- Vesicular Transport
- Vesicles transport molecules between cisternae - Cisternal Maturation
- Cisternae maturate from cis to trans together with cargo molecules
How do other proteins get transported to the cell surface? These other proteins lack signals that ER proteins give off.
- nonselective constitutive secretory pathway transports most other proteins to cell surface
Note:
- No specific signal = secrete the protein (default pathway)
- Specific signals are needed to direct secretory proteins into secretory vesicles and lysosomal proteins into different specialized transport vesicles.
What do you know about nuclear pore complexes (NPCs)?
- mediates what comes in and out of the nucleus
- perforate the nuclear envelope in all eukaryotes
- each NPC is composed of a set of approximately 30 different proteins, or nucleoporins
- unstructured proteins at the inner ring form a mesh (sieve restricting diffusion of large macromolecules, allowing smaller molecules to pass)
What gives proteins the ability to into the nucleus (via a nucleoporin) or exit?
Cytoplasmic proteins with a nuclear localization sequence (NLS) are directed into the nucleus
Notes:
- Nuclear localization signals have flanking basic clusters
- Proteins with a nuclear export sequence (NES) leave the nucleus
What role do importins play in nucleoporins? What complex can it form and then do? How does cargo disassociate?
Importins
- receptors for NLS containing proteins
soluble cytosolic proteins that contain
- multiple low-affinity binding sites for the FG repeats found in the unstructured domains of several nucleoporins.
Complex
- importin–cargo complex locally dissolves the gel-like mesh and can diffuse into and within the NPC pore
Dissociates
- Ran-GTP in the nucleus promotes cargo dissociation
Explain the role of Exportins.
- They are the NES (nuclear export sequence) receptors
- Ran-GTP in the nucleus promotes cargo binding, rather than promoting cargo dissociation as in the case of importins
Explain how GTP hydrolysis of Ran-GDP is activated. What do subunits of importin do? When is importin-beta good to carry another protein? Exportins?
Process: Cytoplasm —> cytosol
Hydrolysis
- a cytosolic GTPase-activating protein (GAP) triggers GTP hydrolysis Ran-GDP, and a nuclear guanine nucleotide exchange factor (GEF) promotes the exchange of GDP for GTP
Subunits
- The α subunit of importin binds the nuclear localization signal
The β subunit binds the unstructured chains.
importin-beta
- In the cytoplasm, a GTP molecule in Ran is hydrolyzed and the Ran dissociates, leaving importin-beta ready to carry the next cargo protein inside
Exportin
- Exportins bind to both the export signal, either directly or via an adaptor, and to NPC proteins to guide their cargo to the cytosol.
Note:
Many proteins are known to have both NESs and NLSs and thus shuttle constantly between the nucleus and the cytosol
Where are mitochondrial proteins synthesized then translocated? What chaperone prevents folding?
- mitochondrial proteins are synthesized in the cytoplasm and then translocated into the mitochondria
- HSP-70 chaperones interact with mitochondrial proteins at the cytoplasm and prevent them from folding
Note:
Mitochondrial and plastid proteins have sequence signals
How are polypeptide chains moved through the two (inner and outer) membranes of the mitochondria?
TIM and TOM systems move polypeptide chains through the two membranes of the mitochondria
Note:
- Translocase of the outer membrane = TOM
- Translocase of the inner membrane = TIM
- For the for chloroplasts : TIC and TOC
What do you know about the cytoskeleton?
- cytoskeleton maintains cell shape, organization, and provides support for internal and external movement
- three classes of cytoskeletal filaments are microfilaments, microtubules, and intermediate filaments
What are microtubules composed of? What’s its structure?
- Microtubules are polymers of the protein tubulin, aka composed of alpha and beta tubulin heterodimers
- microtubule is built from 13 parallel protofilaments (αβ-tubulin heterodimers stacked head to tail and then folded into a tube)
- lattice make them stiff and hard to bend
- are hollow tubes
How do microtubules have polarity? How does it grow?
Polarity
- orientation of their subunits gives microtubules polarity
- the microtubule plus end grows and shrinks much more rapidly than its minus end
Growth
- Rapid microtubule growth occurs by the addition of tubulin dimers at the ends (first lag phase, second elongation phase then lastly plateau phase)
- addition of GTP-tubulin to plus end of a protofilament causes the end to grow in a linear conformation that assembles into the cylindrical wall of the microtubule
Microtubules undergo a process called Dynamic Instability, what does this mean? Catastrophe vs Rescue?
Dynamic Instability
- Individual microtubules alternate between cycles of growth and shrinkage
Catastrophe
- change from growth to shrinkage
Rescue
- change from shrinkage to growth
How does the addition of GTP-tubulin and hydrolysis of GTP affect the microtubules shape?
Addition of GTP-Tubulin
- This addition to the plus end of a protofilament causes the end to grow in a linear conformation that assembles into the cylindrical wall of the microtubule
Hydrolysis of GTP
- occurs after assembly
- changes the conformation of the subunits, forces the protofilament into a curved shape that is less able to pack into the microtubule wall.
Nucleation, the creation of a structure, depends on what complex? What is produced from the nucleation and this complex? Where does this subunit usually reside?
- Nucleation depends on the γ-tubulin ring complex.
- Microtubules are generally nucleated from the microtubule-organizing center (MTOC) where γ-tubulin is most enriched
- Unless the cell is dividing, γ-tubulin is in the centromere
What do you know about centrosomes?
- Its a type of microtubule-organizing center (MTOC)
- composed of two centrioles and surrounded by a dense mass of protein termed the pericentriolar material
- γ-tubulin is in the pericentriolar material
What role do MAPs, Map2 and Tau play in microtubule formation?
- Microtubule associated proteins (MAPs) bind and stabilize microtubules
- Map2 and Tau set the spacing of the microtubule bundles
Note:
- Tau Mutations cause Neurodegenerative Diseases (e.g., Alzheimer’s disease)
What do you know about Kinesin proteins? Describe its structure.
Kinesin
- motors that move towards the plus ends of microtubule
- long-range movement (progressive same size steps, one head is attached to microtubule)
- are apart of a large protein superfamily (common element = motor domain of the heavy chain)
- tetramer protein
- must be inhibited for minus-end transport
Structure
- Two heavy chains and two light chains
- Heavy chain has many domains
- Head splits ATP and converts the energy into motion
- Tail is the cargo-binding
Kinesin-13 proteins (microtubule depolymerases) induces depolymerization from which sides of the microtubule? What else do you know about them?
- Induces depolymerization from both ends of the microtubule.
- are incapable of movement.
- regulate microtubule dynamics to control spindle assembly
Why is kinesin-14 (Ncd) unusual? What does its tail allow it to do?
- unusual as it moves from microtubule plus-ends towards the minus-ends in motility assays
- tail of kinesin-14 can bind microtubules and allows it to organize microtubule bundles
List all that you know about Cytoplasmic Dynein (microtubule motor protein #2).
- Dynein steps are big but irregular and it moves toward the minus ends
- Dynein is ~4 times bigger and more complex than Kinesin
- Head is force generating motor(AAA = ATPase domain)
- stalk contains the microtubule binding site at its tip (so tail binds cargo)
- ATP changes the conformational structure to dissociate microtubule binding
Compare kinesin and dynein.
Kinesin
- Small
- Towards (+) end
- Regular steps
Dynein
- Big
- Towards (-) end
- Irregular steps
Both:
- move vesicles in the secretory pathway
- ATP driven
What are some things you know about cilia and flagella?
- hairlike cell appendages that have a bundle of microtubules & associated proteins at their core (aka the axoneme)
- flagella are found on sperm and many protozoa and have an undulating motion.
- cilia beat with a whiplike motion
- axonemal dynein bends the axoneme which move the cilium and flagellum
Describe & list all that you know about actin filaments (aka microfilaments). How does the way actin subunits assemble provide polarity? Which end does growth occur?
Known:
- helical polymers of the protein actin
actin subunits, (G-actin) are polypeptides carrying a molecule of ATP or ADP
- Actin is an ATPase
- Actin subunits assemble head-to-tail to form a tight, right-handed helix, forming a structure about 8 nm wide called filamentous or F-actin
Polarity:
- At minus end the ATP-binding pocket is exposed
- At the plus end the ATP-binding pocket is buried in the filament
- Growth happens mostly from the + end
Nucleation what step in the formation of Actin Filaments? Describe.
Rate-limiting Step
- two actin molecules bind relatively weakly to each other, but addition of a third actin monomer makes the complex more stable.
- the filament then undergoes a net addition of subunits at the plus end while simultaneously losing subunits from the minus end. (always going to + end)
- actin treadmilling pushes membranes forward
What do actin-binding proteins regulate?
True or false, migrating cells make protrusive structures? If true, name two.
- actin-binding proteins regulate polymerization and length of filaments
- migrating cells make protrusive structures termed filopodia and lamellipodia (crosslinking results in these structures)
Note:
- filopodia are made of actin bundles
- lamellipodium has complex actin networks
What do capping proteins help with? What about Cofilin (any similarities between it and restriction enzymes)? And Alpha-actinin (bundling)/Filamin?
Capping proteins
- prevent G-actin addition and loss
binds to the plus ends
Cofilin (scissors)
- cuts actin filaments into small pieces
A-Actinin
- crosslinking protein that makes F-actin bundles
- it is a dimer & rigid
- crosslinkers must have ≧ 2 actin binding sites
Filamin
- a flexible actin crosslinker protein
crosslinkers must have ≧ 2 actin binding sites
- a dimer
- flexibility allows different angles
We know Actin branching proteins make branches. Describe the Arp ⅔ complex structure and function.
- The Arp2/3 complex promotes actin branching
- Arp2/3 is composed of 7 subunits that are structurally similar to G-actin
- initiates a new branched filament by binding to the side of a filament and recruiting actin monomers.
How is the actin cytoskeleton linked to the membrane?
- actin cytoskeleton is linked to the membrane by ERM proteins
- ERM proteins: Erzin, Radixin, Moesin
Note:
- binding sites for actin and membrane proteins are hidden but revealed when Erzin is phosphorylated
What do you know about small GTPases? GAP & GEF?
- signal the formation of different actin structures
- Ex: Rho Rac and Cdc42
- GTPase activating proteins (GAP) Guanine nucleotide exchange factor proteins (GEF) proteins regulate the activity of the GTPases
Actin-based Motor Proteins Are Members of the Myosin Superfamily. What else do you know about myosin (heads)? How does it walk?
- All myosins share similar motor domains (dark green), but their C-terminal tails (light green) are very diverse. Myosins are conventional or unconventional
- Each myosin head binds and hydrolyzes ATP, using the energy of ATP hydrolysis to walk toward the plus end of an actin filament
Distinguish between Myosin-V, Myosin I and Myosin II, Myosin light chain kinase (MLCK)?
Myosin-V
- two-headed molecule that steps processively along actin filaments, transporting and dispersing cellular cargos in a wide variety of cell types.
Myosin I
- has only one head and binds membranes
- mutants are left/right inverted
Myosin II
- forms bipolar filaments
- composed of two heavy chains and four light chains.
- dimerization occurs when the two α helices of the heavy chains wrap around each other to form a coiled-coil.
MLCK
- regulatory subunits of myosin II
- phosphorylates the myosin light chains and unfolds myosin II into an active state
What are stress fibers, in context of myosin and actin filaments?
Stress fibers are contractile actin bundles found in non-muscle cells. They are composed of actin filaments and non-muscle myosin II.
List the steps of the Myosin power stroke.
- Without ATP myosin is attached to the actin filament in Rigor.
- ATP binding to the myosin head domain causes the release of actin.
- ATP hydrolysis causes a large conformational shift in the ‘lever arm’.
- Associates with actin again.
- Releases the phosphate and does the power stroke, the force-generating step!
- ADP is released myosin head remains tightly bound.
What interactions occur in a skeletal muscle? How are these muscles formed?
- tail–tail interactions form large, bipolar thick filaments that have several hundred myosin heads, oriented in opposite directions at the two ends
- skeletal muscle cells (also called muscle fibers) are big multinucleated cells form by the fusion of many muscle cell precursors
What is the sliding filament theory ~1950?
a muscle fiber contracts when myosin filaments pull actin filaments closer together and thus shorten sarcomeres within a fiber
Notes:
- The A-band (black) does not change
- The I-band (white) disappears
Distinguish between a sarcomere and a myofibril.
Sarcomere
- Each is formed from a miniature, a precisely ordered array of parallel and partly overlapping thin and thick filaments
Myofibril
- is a cylindrical structure 1–2 μm in diameter that is often as long as the muscle cell itself.
- consists of a long, repeated chain of tiny contractile units of sarcomeres
How does sarcomere shortening occur? How would you describe how myosin and actin filaments are packed together?
- sarcomere shortening is caused by the myosin filaments sliding past the actin thin filaments, with no change in the length of either type of filament
- myosin and actin filaments are packed together with almost crystalline regularity
Explain thin and thick filaments in muscle contraction. What holds thin filaments together?
Thin filaments
- are composed of actin and associated proteins, and they are attached at their plus ends to a Z disc (build by Capz) at each end of the sarcomere
- α-actinin holds actin thin filaments together in a regularly spaced bundle
Thick filaments
- are anchored at the M line
Distinguish between Titin and Nebulin in muscle contraction.
Titin
- molecular spring, with a long series of immunoglobulin-like domains that can unfold as stress is applied to the protein.
- keeps the thick filaments poised in the middle of the sarcomere
Nebulin
- maintains the length and the stability of the thin filament
- consists almost entirely of a repeating 35-amino-acid actin-binding motif.
How is muscle contraction initiated? Describe the process.
A Sudden Rise in Cytosolic Ca2+ Concentration Initiates Muscle Contraction
Steps:
1. signal from the nerve triggers an action potential in the muscle cell plasma membrane, and this electrical excitation spreads swiftly into a series of transverse tubules, or T tubules—that extend inward from the plasma membrane around each myofibril.
- When the incoming action potential activates a Ca2+ channel in the T-tubule membrane, it triggers the opening of a Ca2+-release channel in the closely associated sarcoplasmic reticulum membrane
- Ca2+ flooding into the cytosol then initiates the contraction of myofibrils through the Troponin complex and Tropomyosin
- In a resting muscle, the troponin complex pulls the tropomyosin into a position along the actin filament that interferes with the binding of myosin heads.
- When the level of Ca2+ is raised, troponin C—which binds to Ca2+—causes troponin I to release its hold on actin - The increase in Ca2+ concentration is transient because the Ca2+ is rapidly pumped back into the sarcoplasmic reticulum by an ATP-dependent Ca2+-pump
What do epithelial cells establish? Describe.
- establish an apical-basal polarity, which results from the differential distribution of phospholipids, protein complexes, and cytoskeletal components
- within the epithelium, cells are attached to each other directly by cell–cell junctions, where cytoskeletal filaments are anchored, transmitting stresses across the interiors of the cells
What are the three types of junctions? Describe them.
- Tight junctions
- molecules cannot leak freely across the cell sheet (sealed by these junctions)
- visualized by freeze-fracture electron microscopy (branching network of sealing strands encircling the apical end of each cell) - Adherins junctions
- associate with the actin cytoskeleton
- Cadherin/Catenin complex is the main component of the adherent junctions
- cell–cell junction in which cadherin receptors bridge the neighboring plasma membranes via their homophilic interactions - Gap junctions
- clusters of channels that join two cells together and consist of building blocks of two connexons or hemichannels, one contributed by each of the communicating cells
- have a pore size of about 1.4 nm, which allows the exchange of inorganic ions and small molecules, but not of macromolecules
Distinguish between cadherins and catenin (types of adherins junctions).
Catenins
- link classical cadherins to the actin cytoskeleton
- Cytoplasmic protein
- Heterophilic cadherins are called atypical (ex: Fat and Dachsous)
Cadherins
- Aka calcium adherins
- are homophilic (bind to each other)
- transmembrane protein
- cadherin superfamily members all have extracellular portions containing multiple copies of the extracellular cadherin domain
- mediate highly selective recognition, enabling cells of a similar type to stick together and to stay segregated from other types of cells.
Notes:
- Cadherin-Cadherin binding is Ca2+ dependant
- E-cadherin = epithelial
- N-Cadherin = neurona
Describe the cadherin domain.
- Each cadherin domain forms a more-or-less rigid unit, joined to the next cadherin domain by a hinge.
- Ca2+ ions bind to each hinge and prevent it from flexing.
- When Ca2+ is removed, the hinges flex, and the structure becomes floppy
How are strong cell-cell adhesions created?
- Assembly of strong cell–cell adhesions requires changes in the actin cytoskeleton
- Cadherins generate local signals to inhibit the GTPase Rho and activate the GTPase Rac
Freeze-fracture electron microscopy used to visualize tight junctions allows them to appear as sealing strands. What forms these strands? What do insects have instead of tight junctions?
Strands formed by..
- claudins (are transmembrane proteins, related proteins are occludins)
- they form selective channels allowing specific ions to cross the tight-junctional barrier, from one extracellular space to another
Insects
- Tight junctions in insects = septate junctions
- organized differently, but have the same function (also made of claudins)
Describe connexons/hemichannels within Gap junctions. How do invertebrate/vertebrate gap junctions differ with these?
- Each connexon or hemichannel is formed of a complex of six connexin proteins.
- Vertebrate gap junctions are formed by connexins, while invertebrate gap junctions are formed by innexins.
- Both connexin and innexin form similar sized pores
What do you know about the Extracellular Matrix?
- Non-cellular component present within all tissues and organs
- large network of proteins and other molecules that surround, support, and give structure to cells and tissues
- Proteins from the extracellular matrix are secreted (ex: Collagen, proteoglycans, laminin)
- provides essential physical scaffolding for the cell
What are the three proteins that make up the ECM? Describe them.
- Proteoglycans
- have huge oligosaccharide chains
at least one of the sugar side chains of a proteoglycan must be a GAG (Glycosaminoglycan)
Note:
- O-linked glycosylation at the Golgi
- Addition of GAG happens outside the cell
- Fibrous proteins
- polypeptide chains organized approximately in parallel along a single axis, producing long fibers or large sheets
- mechanically strong and resistant to solubilization in water
- Ex: Collagen - Glycoproteins
- contain relatively short oligosaccharide chains
- large scaffold proteins containing multiple copies of specific protein-interaction domains
- have multiple domains and crosslink other matrix molecules and cell receptors
Describe in further detail what you know about one of the ECM proteins GAG (Glycosaminoglycan)? Give an example of the simplest GAG.
GAG
- Aka type of proteoglycans
- are unbranched polysaccharide chains made of repeating disaccharide units (in ECM)
- absorb large quantities of water and swell
Example: Hyaluronan (also called hyaluronic acid or hyaluronate)
- the simplest of the GAGs
- made out directly from the cell surface by an enzyme complex embedded in the plasma membrane.
- Role = space filler and compression protection
Describe in further detail Collagen, a type of fibrous protein in the EMC.
Collagen
- the molecule is along, stiff, triple stranded helical structure, in which three collagen polypeptide chains, called α chains.
- many molecules assemble at the ECM into thick long Collagen fibers
- collagen fibrils form structures that resist stretching forces
- Not all collagens form giant fibrils, some are fibril-associated collagens
Note:
- Defects in the structure or processing of the protein collagen affect the elasticity of the connective tissue
Describe in further detail Fibronectin and Laminin, a type of glycoprotein in the EMC.
Fibronectin
- a dimer joined by disulfide bonds
Laminin
- large glycoprotein composed of three chains and links the ECM to cell surface receptors
Describe the role Integrins play in the ECM, how they link and their structure.
Role
- They are transmembrane heterodimers that link ECM to actin cytoskeleton
- cluster to form strong and dynamic connections to the ECM called Focal adhesion sites
- Epithelia without integrins detach from the basal lamina
How they link
- The extracellular portions of integrin bind ECM proteins like fibronectin or collagen
- The intracellular integrin tails bind to a complex of adaptor proteins that link the actin cytoskeleton (eg, Talin and Vinculin)
Structure
- Two integrin conformations (inactive=binding sites hidden and active=binding sites shown)
- Integrins are dimers
- The cytoplasmic region binds talin
Activation
- can be activated from the outside or from the inside fibronectin peptide talin
- Ie: Binding to a ECM substrate or binding to talin in the inside
Integrins create focal adhesion sites when they cluster, describing this process. There are two main components to it, name them.
Focal adhesions
- are large macromolecular assemblies that form mechanical links between intracellular actin bundles and the ECM
- All the mechanical signaling makes focal adhesions very dynamic
Talin and Vinculin (main components)
- Talin is an elastic scaffold protein and a tension sensor at cell–matrix junctions
- Talin brings a protein called focal adhesion kinase (FAK) to the focal adhesion
- Talin has a large number of binding sites for the actin-regulatory protein vinculin. Most sites are hidden inside folded protein domains but are exposed when those domains are unfolded
- Vinculin stabilizes talin, actin filaments, and integrins
- Vinculin opens up and aids integrin clustering for FA growth, has open and closed conformations
Epithelial cells establish an apical-basal polarity, how does this happen? What do antagonist interactions aid with?
- apical-basal polarity results from the differential distribution of phospholipids, protein complexes, and cytoskeletal components between the various plasma membrane domains, reflecting their specialized functions.
- Antagonistic interactions between polarity factors that maintain the identity and control the size of the apical, junctional and lateral domains
Apicobasal polarity depends on the action of three protein complexes. Name them.
- The Par Complex
- cytoplasmic - The Crumbs Complex
- only transmembrane protein and links aPKC to the membrane - The Scribble Complex.
- Cytoplasmic
- inhibits aPKC
Notes:
- They are mutually inhibitory
- The exact position of the complexes vary slightly between tissues and species.
Why is aPKC is the main effector of apical identity? What does the phosphorylation of Lgl inhibit?
aPKC
- main effector of apical identity because it phosphorylates the junctional and lateral polarity factors Par-3 and Lethal (2) giant larvae (Lgl) to exclude them from the apical domain
Phosphorylation of Lgl inhibits its association with the other proteins in the complex
- aPKC, a kinase, phosphorylates and inactivates Lgl
Using flies as an example, explain the use of an “imaginal disc” in terms of polarity & tumors.
- An imaginal disc is a sac-like epithelial structure found inside the larva of insects that undergo metamorphosis.
- Without AP polarity the discs overproliferate and form tumors (AP polarity and cancer are linked)
What does the loss of apicobasal polarity lead to? Explain EMT.
Loss
- the loss of apicobasal polarity leads to
overproliferation and metastasis
EMT
- The epithelial–mesenchymal transition (EMT) is a process by which epithelial cells lose their cell polarity and cell–cell adhesion, and gain migratory and invasive properties
Note:
- tumor suppressors are genes that when mutated lead to uncontrolled proliferation
What is planar cell polarity (PCP)?
PCP
- coordinated polarization of cells within the plane of a tissue
- Ie: Cell orient to the tissue axis, each trichome comes from a single cell
controls hair orientation (mutants have disorganized cell structures (eg. trichomes)
- regulate trichome positioning via changes in actin cytoskeletal organization
Notes:
- Z = AB polarity
- X+Y is Planar cell polarity
What do core proteins self-organize into? Why?
What do actin-binding proteins restrict the formation of? Why?
Core proteins
- The core proteins self-organize into mutually antagonistic sides
- The cytoplasmic proteins mediate the positive and negative interactions
Actin-binding proteins
- restrict formation of the actin-rich trichome to one side
- overall logic is to separate actin processes in the distal and proximal side
PCP is controlled by two main pathways, name and describe both.
- Core pathway
- comprises six distinct proteins that form asymmetrically localized intercellular complexes (ex: Distal and proximal complexes) - Ds-Ft pathway
- aligns the entire tissue - “global pathway”
- Ft and Ds are atypical cadherins
- They form heterophilic complexes between adjacent cells, and localize asymmetrically
- Asymmetric localization of Ft and Ds is driven by expression gradient of Fj and Ds
Notes:
- In the Golgi, Fj phosphorylates extracellular cadherin repeats of Ft and Ds.
- Modifies Ft-Ds binding affinities
Explain what you know about fluorescence. How does this correlate to absorption/emission of photons (e-)?
Fluorescence
- It is the emission of light by a substance that has absorbed light or other radiation
- Fluorescent molecules absorb light at one wavelength and emit it at another, longer wavelength
Photons (or e-)
- An orbital electron of a fluorochrome molecule can be raised to an excited state after the absorption of a photon.
- Fluorescence occurs when the electron returns to its ground state and emits a photon of light at a longer wavelength
- Too much exposure to light destroys the fluorochrome molecule in a process called photobleaching
Explain the use of immunofluorescence.
Immunofluorescence is commonly used in molecular and cell biology labs as a robust and simple method to reliably localize molecules on fixed cells or tissues
Where does the original green fluorescent protein originate from? How does it produce luminescence? List all that you know about it (ex: does it need a prosthetic group, how are proteins visualized).
Origin
- original green fluorescent protein comes from a jellyfish (Aequorea victoria)
Luminescence
- Aequorin is a blue-light-emitting bioluminescent protein.
- GFP is a green-light emitting protein (absorbs blue, emits green)
- Aequorin and GFP work together to convert Ca2+-induced luminescent signals into the green luminescence
More info
- GFP does not need a prosthetic group or any other cofactor so it can be entirely genetically encoded. The chromophore is made by amino acids
- Fusing GFP to the coding sequence of a gene allows direct visualization of the protein
- peptide location signal can be added to the GFP to direct it to a particular cell compartment, such as the endoplasmic reticulum or a mitochondrion
- GFP fluorescence responds rapidly and reversibly to pH changes
What do mutations in GFP (green fluorescent protein) result in?
- Mutations in GFP change the absorption and emission colors
- also change other fluorescence properties, like brightness stability, maturation time, etc
What is an experiment we can do with fluorescent proteins that we couldn’t do before? Can we tell the age of proteins?
Experiment: Track the movement of proteins and cells in-vivo
- Visualizing intracellular Ca2+ concentrations by using a fluorescent indicator
- calcium imaging is a powerful means for monitoring the activity of distinct neurons in brain tissue in vivo (changes in calcium = neuron activity)
Age
- Some fluorescent proteins slowly change their color so we can test the “age” of proteins and cells
Compare photobleaching and FRAP?
Photobleaching
- photochemical alteration of a fluorophore, that makes it unable to fluoresce.
FRAP
- Means fluorescence recovery after photobleaching
- Indicates dynamics of a protein in a living cell (ie: how fast proteins move)
What technique do we use to gauge the distance between two chromophores? Can GFP do this as well? Explain.
Technique:
- Fluorescence Resonance Energy Transfer (FRET) is a special technique to gauge the distance between two chromophores
- FRET can be used to make Genetically encoded fluorescent biosensors many molecular sensors
GFP
- Split GFP proteins into fragments that spontaneously assemble into a functional protein when in very close proximity
Notes:
- In proximity = FRET
- Not in proximity = No FRET
- Test protein to protein binding (1–10 nm)
What is hybridization?
Hybridization is the process of combining two complementary single-stranded DNA or RNA molecules and allowing them to form a single double-stranded molecule through base pairing.
What can RNA hybridization do? What is the difference between injecting dsRNA vs dsDNA into worms and the effect passed to progeny?
RNA hybridization
- allows detection of mRNA molecules
- For example: uses in target hybridization and fluorescence imaging
dsRNA
- has a strong effect
- Once injected and passed onto offspring, the worm twitches in a similar way as if it had defective muscle protein gene
dsDNA
- inhibits synthesis of specific proteins
promotes mRNA degradation
- can be introduced experimentally to silence genes of interest
RNA interference (RNAi) is a mechanism of gene regulation. Explain the steps in how it is accomplished.
- Long double-stranded RNA (dsRNA) is cleaved into small interfering RNA (siRNA) by the enzyme Dicer.
- The siRNA joins the RNA-induced silencing complex (RISC),
- Argonaute 2 (AGO2) cleaves the sense strand of RNA .
- The RISC–siRNA complex binds and degrades complementary mRNA
- The activated RISC–siRNA complex can then be reused
What else do you know about RNAi? What are two possible applications? Could this cure a well-known neurodegenerative disease?
other RNAi info
- RNA interference is a defense against viruses and jumping genes especially crucial for plants, worms, and insects
allows remove specific proteins in cells and tissues without affecting the genome
Applications
1. Therapeutic
- Remove malfunctioning proteins that cause problems
- Destroy viruses
- Research
- Study the functions of individual proteins
Cure Huntington’s Disease (??)
- Huntington’s is caused by mutations in huntingtin gene
- The mutated huntingtin proteins accumulate in neurons and cause brain damage
How would you use an RNAi screen in cells to identify cell morphology proteins?
- Identify an interesting process → e.g., cell morphology
- Get a library of dsRNA that cover all the genes
- Incubate cells with specific dsRNA
score the changes
