chapter 9,11, 12, 13 Flashcards
how are antibodies used to visualize specific proteins within cells and tissues
- primary antibody binds to target protein (antigen A); secondary antibody (marker-coupled) recognizes primary antibody
- immunofluorescence; immunohistochemistry; western blotting
allows us to amplify signal because proteins can be very small. Multiple secondary antibodies can recognize a primary antibody to amplify signal
What is fluorescence
a physical property of an object absorbing light at one wavelength and then reemitting it at another
single molecule localization microscopy
- to determine the precise localization of specific molecules
- stimulated emission depletion microscopy
what does EM tomography do?
- to study larger structures with same resolution as SEM
- specimen tilted to maximum of 60° to allow imagingn form multiple angles and give 3D reconstruction
TEM sample prep
glutaraldehyde- fixative that cross-links proteins
osmium tetroxide- binds and stabilizes lipid bilayers and proteins
uranium and lead- staining tissue to visualize specific cell components
tissue stained with electron-dense materials, which adhere to the cells but dont target a specific protein
cryo-EM
- specimen is rapidly frozen and imaged of all orientations without further processings
- can determine structure of macromoecules without need to crystalize
What are 2 ways to color cells using light microscope? And what is it for?
- Chromogenic dyes
- Fluorescent dyes
to visualize cell compartments
rank ease of passage of molecules through the membrane
- hydrophobic molecules (gases, steroid hormones)
- small uncharged polar molecules (H2O, urea, glycerol, NH3)
- large uncharged polar molecules (glucose, sucrose)
- ions
channel vs transporter
channel- allow specific solutes to pass; faster transport rate; weaker binding to affinity to solutes; passive only
transporter- bind to solute and undergo conformational changes; may be passive or active (pumps)
Sarcoplasmic reticulum
type of ER found in muscle cells
active transport molecules
coupled transporter- using favorable gradient to drive unfavorable gradient
ATP-driven pump- ATP hydrolysis to power transport of something against its gradient
light-driven pump- light or redox energy to power transport against gradient (in bacteria, mitochondria, chloroplast)
active or passive?
uniport
symport
antiport
both
uniport- moving one solute in either direction
symport- moving 2 solutes in same direction (coupled)
antiport- moving 2 solutes in different direction (exchange) (coupled)
what type of transport is glucose transport from gut lumen to EC fluid?
passive simport because Na+ naturally wants to exit
Vmax and Km
Vmax- rate at which transporter flips between conformational states
Km- affinitity to solute
gating mechanisms for ion channels
- volatage-gated
- ligand-gated- extracellular ligand
- ligand gated- intracellular ligand
- mechanically gated
always passive
How is an action potential initiated and terminated at a nerve terminus?
Initiated: voltage-gated calcium channels to open, allowing calcium ions to influx and trigger the release of neurotransmitters from synaptic vesicles
terminated :by the inactivation of sodium channels and the opening of potassium channels, which repolarizes
- Identify the primary ions and channels involved in each phase of an action potential
Role of inactivation in the action potential. What happens if channels do not inactivate?
Inactivation ensures that once an action potential has been initiated, the neuron cannot be continuously depolarized by the influx of Na⁺.
sustained influx of sodium ions, causing prolonged depolarization of the neuron. This could prevent the membrane potential from returning to its resting state.
saltatory conduction
action potential propagates along myelinated axon jumping from node to node (just from one node of ranvier to another)
What can action potential not cross?
a synapse
schwann cells vs oligodendrocytes
schwan cells- myelinated axons in PNS
oligodendrocytes- myelinated axons in brain
how can an action potential spread along distances?
by depolarizing neighboring regions of the membrane
what controls direction of action potential propagation?
refractory period (Na channels are innactivated, so action potential is pushed to where they are closed)
how can some molecules be at equilibrium, across a membrane but not be the same concentration on both sides?
electrochemical gradient
nucleolus function
rRNA transcription and ribosome assembly
stress granules functions
- temporary storage, particularly of translation-related components
- formed in response to specific stress; contain mRNA
p-granules functions
RNA metabolism and inheritance
postsynaptic density function
organization of macromolecules in dendrites needed for neuronal transmission
difference between a membrane bound organelle and a biomolecular condensate
condensates- membraneless organelles; formed from multiple weak interactions between proteins and macromolecules; subcellular organizations where proteins/nucleic acids are assembled into a functional unit; can form and dissolve PRN
Explain liquid-liquid phase separation
- property of biomolecular condensate (membraneless organelles)
- remains liquid but does not dissolve in the surrounding cytosol
describe how the topology of intracellular compartments has evolved
rough vs smooth ER functions
rough- translation of ER-destined proteins
smooth- sites for budding and fusing of transport vesicles
describe the mechanism that inserts proteins in the endoplasmic reticulum, for both soluble and
transmembrane proteins, naming the key components
what 2 kinds of proteins are transferred from cytosol to ER?
- soluble: transferred completly to the Er lumen; destined for secretion out of cell or lumen of another organelle (not mitochondrion or plastid)
- transmembrane: partly dislocated into ER, embedded in membrane; destined to reside in ER or membrane of another organelle or plasma membrane
both initally directed to ER by ER signal sequence
membrane-bound vs free ribosomes
membrane-bound: makes proteins that are being translocated to the ER
free: make all other proteins
ER signal sequence vs ER retention sequence
signal: N-terminal stretch of hydrophobic amino acids
retention: C-terminus (KDEL amino acids)
Explain how ER-designated proteins are recognized and transported
Modifications that proteins undergo in the ER
formation of disulfide bonds- stabilize protein structure, only for proteins that are going to be secreted or have extracellular facing domain; oxidation of cystein
glycosylation - carbohydrate layer for proteins that face extracellular side; aids with folding of proteins such as in case of calnexin
Sec61 complex
translocator protein that inserts protein into ER
What does accumulation of misfolded proteins in ER trigger?
UPR to make more ER to make more chaperones
if misfolded proteins continue to accumulate and ER has reached full capacity, UPR directs cell death by apoptosis
What happens if protein fails to fold in ER?
protein exported to cytosol and degraded by proteasomes
Explain the molecular basis for cystic fibrosis
mutation in chloride transporter channel causes slight misfolding, which causes protein retention in the ER and degradation. Otherwise, the protein could be functional if reached the membrane
disease is because channel cant fold it property
Compare and contrast the transport to mitochondria and chloroplast
mitochondria primarily relying on an electrochemical proton gradient across their inner membrane while chloroplasts utilize ATP and GTP hydrolysis for import into their stroma and thylakoid lumen respectively
What complexes move proteins across outer and inner mitochondrial membrane?
Outer: MIM1, TOM, SAM
Inner: TIM22, TIM23, OXA
how are mitochondrial proteins transported?
as unfolded polypeptide chains
Name different compartments of mitochondria and chloroplasts and describe how proteins can be transported into each of the compartments
Describe the nuclear pore
- “gates” in membranes of the nucleus that allow movement of molecules in both directions
- small molecules can freely diffuse through nuclear pore but large molecules like proteins and RNA cannot pass without appropriate sorting signal/modificaitons
Signal sequences and their function
- needed to pass through nuclear pore
- short stretch of lysines/arganinines (positive charges)
importins vs exportins
importins: nuclear import receptors; recognize nuclear localization signal and facilitate transfer of cargo into nucleus
exportins: nuclear export receptors; facilitate transfer of cargo out of the nucleus
Diagram the Ran GTP/GDP cycle
How are nuclear import and export similar?
Ran GTP increases affinity to cargo when export
changing the concentrations changes the direction of transport
Compare and contrast the different transport systems between ER, mitochondria, chloroplast and nucleusx
List the different secretory pathways and give example of secretory cells
COP2 coated vescicles: package and move collagen fibers (ER –> golgi)
COP1: golgi –> ER
clathrin: princh from plasma membrane
Diagram protein movement in the Golgi
- Cis –> trans
- proteins travel through cisternae by vesicular transportor by golgi cisternae themselves which migrate thourhg staacks
- forward pathway: ER to golgi
Explain the difference between phagocytosis and pinocytosis
phagocytosis: cellular eating; occurs in specialized cells that digest large molecules
pinocytosis: ingestion of fluid and small molecules; all eukaryotic cells all of the time
Explain receptor-mediated endocytosis, for instance of cholesterol
phosphorylation to turn on/off transport signals
binding to anchoring proteins that localize compartments
1. cell bound to SREBP when dont need cholesterol. When found in environment because high cholesterol.
2. packaged into vesicle and sent to golgi
3. released transcription regulatory protein into cytoplasm
4. goes into nucleus and makes cholesterol biosynthesis enzymes
Explain the difference between endosomes, late endosomes, and lysosomes
endosomes- large network of membrane tubes and vesicles that sort molecules
early endosomes- just beneath plasma membrane; pH 7
late endosomes- closer to the nucleus; lower pH; involved in final stages of sorting and processing
lysosomes- contain hydrolytic enzymes that digest macromolecules; lumen is acidic for enzymes
autophagy vs mitophagy
autophagy- digestion of organelles by cell itself (signal: mTOR complex)
mitophagy- selective destruction of mitochondria (PINK1 and parking promote ubiquinations)
What is topologically equivalent to extracellular space?
ER lumen
What organelle has similar topology to cytosol?
nucleus
Long-term potentiation vs long term depression
LTP: strong stimulation of a postsynaptic neuron by a specific presynaptic neuron will generate reaction to subsequent weak stimulations, increase in synaptic length between 2 neurons
LTD: result in loss of synapse between neurons
Pathways that directly deliver materials to lysosomes
- Endocytosis
- Autophagy
- Macropinocytosis
- Phagocytosis
