Cell Bio Quiz Week 4 Flashcards
Why do we use fixatives in microscopy?
Fixatives preserve cellular structures by cross-linking(using fixatives to form bonds between proteins) proteins to prevent degradation. Glutaraldehyde is commonly used but toxic; formaldehyde is an alternative.
Flashcard 2: What are the key steps in preparing a sample for microscopy?
- Fixation – Preserves structure.
- Embedding – Supports thin slicing (paraffin for LM, resin for EM).
- Sectioning – Using a microtome to create thin slices.
- Staining – Enhances contrast (H&E, DAPI, immunostaining).
How does fluorescence microscopy work?
Uses fluorescent dyes that emit light when excited by a specific wavelength. This allows visualization of specific proteins or organelles.
What is the difference between TEM and SEM?
TEM (Transmission Electron Microscopy): Produces 2D images of internal structures.
SEM (Scanning Electron Microscopy): Produces 3D images of the surface.
How do we measure membrane fluidity?
Fluorescence Recovery After Photobleaching (FRAP): A laser bleaches a membrane area, and recovery speed of fluorescence measures membrane fluidity.
What are the three types of cell surface receptors?
Ion-channel-linked receptors – Open/close ion channels.
G-protein-coupled receptors (GPCRs) – Activate G proteins, triggering cascades.
Enzyme-linked receptors – Direct enzymatic activity (e.g., tyrosine kinases).
How do GTP-binding proteins function in signaling?
Active when bound to GTP, inactive with GDP.
Two types:
1. Heterotrimeric G proteins (used in GPCR signaling).
2. Monomeric GTPases (e.g., Ras, involved in cancer).
What is positive vs. negative feedback in signaling?
Positive feedback: Amplifies signals (e.g., blood clotting).
Negative feedback: Suppresses signals (e.g., insulin reduces glucose).
What is mitochondrial fission and fusion? (and what regulates it)
Fission: Splitting mitochondria (removes damaged parts).
Fusion: Merging mitochondria (allows exchange of mtDNA and proteins).
Regulated by GTPases:
- change in dnm (fission).
- change in fzo1(fusion).
What is the function of StAR protein?
Steroidogenic Acute Regulatory Protein (StAR) moves cholesterol from the outer to the inner mitochondrial membrane for steroid synthesis.
How is cholesterol metabolized for steroid production?
Uptake: via LDL receptors and stored in endosomes/lysosomes.
Transported to mitochondria: by StAR.
Converted into pregnenolone: by P450scc (CYP11A1)
What diseases result from disrupted cholesterol transport?
Congenital adrenal hyperplasia (CAH) → Deficiency in steroid hormones.
Salt and carbohydrate imbalance.
Ambiguous genitalia in newborns due to hormone disruption.
What is the structure of GPCRs?
Seven-pass transmembrane receptor.
Extracellular domain binds ligands (hormones, neurotransmitters).
Intracellular domain interacts with G proteins.
How does GPCR activation work?
Ligand binds to GPCR.
GPCR changes shape and binds inactive G-protein (GDP-bound).
GDP is swapped for GTP, activating the G-protein.
Gα separates and activates downstream pathways.
What are the two major GPCR signaling pathways?
Some activated G
proteins work via
increasing the
amount of cAMP
- cAMP Pathway (Adenylate Cyclase Activation)
- Gαs activates adenylate cyclase → increases cAMP.
- cAMP activates PKA, which phosphorylates proteins.
- Phospholipase C (PLC) Pathway
- activates PLC, which cleaves PIP2 into IP3 and DAG.
- IP3 releases Ca²⁺ from the ER.
- DAG activates PKC, leading to cellular responses.
- Example: Vasopressin → GPCR → PLC → Water reabsorption in kidneys.
How are small GTPases (monomeric G proteins) regulated?
GEFs (Guanine nucleotide exchange factors) activate them (GDP → GTP).
GAPs (GTPase-activating proteins) turn them off (GTP → GDP).
If monomeric G potein is activated, it can initiate downstream signaling cascades. One of the well-known pathways is the MAPK cascade (this happens when there is activation of monomeric G protein, Ras)
How was it discovered that mitochondria contain GTP-binding proteins?
Before 1995, GTP-binding proteins were not thought to exist in mitochondria.
Western blotting with radioactive GTP revealed their presence.
Why are GPCRs important?
GPCRs regulate vision, taste, smell, neurotransmission, hormone signaling, and immune response.
Target of ~50% of all drugs, including beta-blockers and antihistamines.
What is the function of phospholipase C (PLC)?
Phospholipase C (PLC) is an enzyme that cleaves PIP2 (phosphatidylinositol 4,5-bisphosphate) into two second messengers:
- IP3 (inositol 1,4,5-triphosphate) → Triggers Ca²⁺ release from the ER.
- DAG (diacylglycerol) → Stays in the membrane and activates Protein Kinase C (PKC).
How is PLC activated?
Phospholipase C (PLC) is an enzyme that plays a key role in cell signaling by breaking down membrane phospholipids to generate second messengers.
PLC is activated by G-protein-coupled receptors (GPCRs) and Receptor Tyrosine Kinases (RTKs) through different mechanisms
GPCR
*Ligand Binds to GPCR causing its α-subunit to exchange GDP for GTP.
*PLC Activation: *The activated subunit binds to and activates PLC.
Breakdown of PIP₂: PLC hydrolyzes (PIP₂) into two second messengers:
*Inositol trisphosphate (IP₃) *– Triggers calcium (Ca²⁺) release from the endoplasmic reticulum.
Diacylglycerol (DAG) – Activates protein kinase C (PKC), leading to further signaling.
What are the roles of IP3 and DAG?
IP3:
- Water-soluble, diffuses through cytoplasm.
- Binds to IP3 receptors on the ER, triggering Ca²⁺ release into the cytoplasm.
DAG:
- Stays in the plasma membrane.
- Activates Protein Kinase C (PKC), leading to phosphorylation of target proteins.
How do CRH and Vasopressin stimulate ACTH release?
Corticotropin-releasing hormone (CRH) and Vasopressin (VP) regulate adrenocorticotropic hormone (ACTH) release from the pituitary.
They use different second messenger pathways:
- CRH → Activates the cAMP pathway.
- Vasopressin → Activates the IP3/Ca²⁺ pathway.
ACTH stimulates cortisol release from the adrenal gland, part of the stress response.
What is a superfusion chamber, and why is it used?
A superfusion chamber allows real-time measurement of hormone release and second messenger activity.
Why use it?
- Helps study how cells respond to hormones over time.
- Differentiates fast and slow responses in cell signaling.
How does forskolin affect ACTH and β-endorphin release?
Forskolin is a natural compound that activates adenylyl cyclase, leading to an increase in cyclic AMP (cAMP) levels
Higher cAMP levels enhance ACTH and β-endorphin release (CRH pathway).
Demonstrates that CRH and Vasopressin use different signaling pathways.
What are the effects of phorbol esters on ACTH release?
Phorbol esters activate PKC, mimicking DAG signaling. This causes increased phosphorolation and causes increased ACTH and β-endorphin release via the IP3/DAG pathway (Vasopressin signaling pathway).
Key experiment: Showed that CRH and Vasopressin work through separate pathways.
What happens during a calcium wave in a fertilized oocyte?
Sperm entry triggers Ca²⁺ release from the ER, spreading as a wave.
Why important?
- Initiates oocyte activation.
- Ensures proper embryo development.
- build barrier so no more can enter?
How is calcium stored in the cell?
Stored in:
- Endoplasmic Reticulum (ER) – when released, it is done by IP3 receptors.
- Mitochondria – Stores Ca²⁺ using the mitochondrial calcium uniporter (MCU).
What causes calcium oscillations?
Positive feedback amplifies Ca²⁺ release, creating oscillations.
Negative feedback restores balance.
Example: Vasopressin-induced Ca²⁺ waves in liver cells.
What is the function of calmodulin?
Calmodulin (CaM) is a Ca²⁺-binding protein that regulates many cellular processes.
When Ca²⁺ binds to calmodulin, it activates target enzymes like CaM-Kinase (CaMK).
How does CaM-Kinase work?
CaM-Kinase is activated in a stepwise manner:
1. Ca²⁺ binds to calmodulin.
2. Calmodulin activates CaM-Kinase.
3. CaM-Kinase phosphorylates itself, increasing activity.
4. Has “molecular memory” → Remains active even after Ca²⁺ levels drop.
How do fluorescent calcium sensors work?
Dyes like Fura-2 bind to Ca²⁺ and change fluorescence intensity, allowing real-time tracking of Ca²⁺ dynamics.
How is calcium involved in muscle contraction?
Nerve stimulation releases Ca²⁺ from the sarcoplasmic reticulum (SR).
Ca²⁺ binds to troponin, exposing binding sites on actin.
Myosin binds actin, causing contraction.
Ca²⁺ is pumped back into the SR to relax the muscle.
Why is there a lag phase in muscle contraction?
The calcium release channels take time to open, leading to a short delay before contraction begins.
What are the main differences between CRH and Vasopressin signaling?
** CRH Pathway
**
- GPCR (Gs) (receptor)
- cAMP(second messenger)
- activates PKA
- ACTH is released via cAMP stimualtion
** Vasopressin Pathway**
- GPCR (Gq) (receptor)
- IP3 & DAG (second messengers)
- releases Ca²⁺, Activates PKC
- ACTH release via Ca²⁺ stimulation
Why is calcium signaling important?
wer:
Regulates hormone secretion, muscle contraction, neurotransmission, fertilization, and metabolism.
Highly controlled by pumps, channels, and feedback mechanisms.
What are enzyme-coupled receptors?
Transmembrane receptors that trigger intracellular enzyme activity when a ligand binds.
The largest family is Receptor Tyrosine Kinases (RTKs).
What are receptor tyrosine kinases (RTKs)?
Enzyme-coupled receptors that regulate growth, survival, and metabolism.
Activated by growth factors like:
- Epidermal Growth Factor (EGF) → Stimulates cell proliferation & differentiation.
- Insulin-Like Growth Factor (IGF) → Important for growth & metabolism.
How do RTKs become activated?
Ligand binds, causing two RTKs to dimerize.
Autophosphorylation – Each RTK phosphorylates the other on tyrosine residues.
Phosphotyrosines act as docking sites, recruiting intracellular signaling proteins.
What is the MAPK cascade?
Ras activates Raf (MAPKKK).
Raf phosphorylates MEK (MAPKK).
MEK phosphorylates ERK (MAPK).
ERK enters the nucleus, regulating gene transcription for cell proliferation.
How can we measure Ras activation in real-time?
Fluorescence Resonance Energy Transfer (FRET) is used.
How?
- Cells are engineered to express Ras fused to a fluorescent protein (YFP).
- Fluorescence changes when Ras is activated, confirming real-time activation.
What is PI3-Kinase and why is it important?
PI3-Kinase (PI3K) is activated by RTKs and generates PIP3 in the membrane.
PIP3 acts as a docking site for proteins involved in:
- Cell survival (Akt/PKB activation).
- Metabolism (insulin signaling).
How does α-Interferon trigger antiviral responses?
α-Interferon blocks viral infection in cells by triggering
resistance proteins
α-Interferon binds its receptor, activating the JAK-STAT pathway.
Triggers resistance proteins, blocking viral infection.
Similar pathway used by:
- Prolactin.
- Erythropoietin (EPO).
- Growth Hormone.
How does the JAK-STAT pathway work?
α-Interferon binds its receptor, causing dimerization.
JAK (Janus Kinase) phosphorylates the receptor.
with receptor phoshphylated, STAT (Signal Transducer and Activator of Transcription) binds to the receptor.
when binding, STAT is phosphorylated and dimerizes.
STAT dimer enters the nucleus, activating gene transcription.
What is the function of TGF-β (Transforming Growth Factor-Beta)?
A superfamily of cytokines regulating:
- Development.
- Wound healing.
- extracellular matrix
- biological functions
etc.
How does TGF-β activate Smad signaling?
TGF-β binds its receptor, causing dimerization.
Receptor phosphorylates Smad proteins.
Smad proteins form a complex and enter the nucleus.
Regulate genes for extracellular matrix production & cell differentiation.
study microscope
do it
two waves in sync
creates brighter
two waves out of sync
make it dimmer
General pathway of intracellular messenger systems
extracellular signal molecule binds to receptor protein. moves to intracellular signalling proteins and later moves to effector proteins
effector proteins
metabolic enzyme- altered metabolism
gene regulatory protein- altered gene expression
cytoskeletal protein- altered cell shape or movement
different forms of intracellular signaling
Look at the graphics
contact dependent
paracrine
endocrine
synaptic
cell surface receptors
intracellular receptors
Fast vs slow response
Some signals cause immediate effects, while others take longer:
Fast responses: Modify existing proteins (e.g., opening ion channels).
Slow responses: Involve gene transcription, leading to long-term effects (e.g., cell growth, differentiation).
Modular Interaction Domains in Signalling Proteins
Signaling proteins have specialized domains that allow them to recognize and bind specific molecules.
Example domains:
- SH2 domains (bind phosphorylated tyrosines).
- PH domains (bind phospholipids in membranes).
- PDZ domains (involved in protein-protein interactions).
Desensitisation of Target Cells
Negative feedback
Delayed feed-forward
Receptor inactivation
Receptor sequestration
Receptor destruction
HPA Axis
The HPA axis is an example of endocrine signaling:
- The hypothalamus releases CRH (corticotropin-releasing hormone).
- The pituitary releases ACTH (adrenocorticotropic hormone).
- The adrenal glands release cortisol, which regulates metabolism and immune response.
This system uses negative feedback to maintain balance.
rate-limiting step in steroid synthesis
Transport of cholesterol from the outer to inner mitochondrial membrane. Cholesterol is hydrophobic and cannot freely diffuse across the mitochondrial membranes.
Cholesterol is delivered to the outer mitochondrial membrane by carrier proteins such as Steroidogenic Acute Regulatory Protein (StAR).
StAR facilitates cholesterol transfer across the intermembrane space to reach the inner membrane.
Cholesterol reaches the inner membrane, where the enzyme P450scc (CYP11A1, also called cholesterol side-chain cleavage enzyme) converts cholesterol into pregnenolone, the precursor to all steroid hormones.
“Labile protein”
The experiment shown in the graph suggests that steroidogenesis requires this labile protein to function properly.
Interrupting protein synthesis with inhibitors like puromycin or cycloheximide inhibits pregnenolone synthesis, indicating that the labile protein is essential for cholesterol transport into mitochondria.
This labile protein was later identified as StAR (Steroidogenic Acute Regulatory Protein).
Contact Site
Contact sites are regions where outer and inner mitochondrial membranes interact.
These sites contain specific proteins that help transfer cholesterol for steroid synthesis.
Protein Kinase A (PKA) in Mitochondria
PKA (cAMP-dependent protein kinase) is found in steroidogenic mitochondria.
PKA regulates StAR activation and cholesterol movement.
Discovery of PKA anchoring proteins (2012) revealed how PKA is localized in mitochondria.
Inactive G protein structure
In the resting state, the G-protein is bound to GDP (Guanosine Diphosphate).
Three subunits (α, β, γ) are together, attached to the membrane.
The receptor is also inactive until a ligand binds.
cAMP pathway
Some G-proteins activate adenylate cyclase, an enzyme that produces cyclic AMP (cAMP).
cAMP is a second messenger that activates Protein Kinase A (PKA).
PKA phosphorylates proteins, leading to various cellular responses.
PLC pathway
Some G-proteins activate Phospholipase C (PLC).
PLC cleaves a membrane lipid (PIP2) into:
IP3 (Inositol triphosphate) – Releases Ca²⁺ from the ER.
DAG (Diacylglycerol) – Activates Protein Kinase C (PKC).
western blotting to detect G proteins
Western blotting uses antibodies to detect proteins in a sample.
In this experiment:
Blot is exposed to radioactive GTP, which binds G-proteins.
Results showed G-proteins in mitochondria!
phospholypase enzymes
hydrolyze phospholipids, releasing secondary messengers that regulate intracellular signaling.
calcium
is a universal second messenger involved in muscle contraction, neurotransmitter release, and gene expression.
synthesis of PIP2
PIP2 is a membrane phospholipid precursor for second messengers.
Synthesized from:
Phosphatidylinositol (PI) → Phosphatidylinositol 4-phosphate (PI4P) → PIP2.
Important for:
Phospholipase C (PLC) signaling.
Membrane trafficking and cytoskeleton organization.
Know conversions
micro- u- 10^-6
nano- n - 10^-9
pico- p- 10^-12
Contact dependent signalling
signaling molecules remain attached to the signaling cell’s surface and require direct cell-to-cell contact for signal transmission.
paracrine signalling
signaling cell releases molecules that diffuse locally to affect nearby target cell
synaptic signalling
Rapid, neuron-specific signaling
endocrine- Long-distance via hormones in the bloodstream (e.g., insulin)
Cell surface receptor signalling
extracellular signals (e.g., hormones, growtth factors, cytokines) bind to specific receptors on the plasma membrane, triggering intracellular responses. Since many signaling molecules are hydrophilic and cannot cross the lipid bilayer, they rely on membrane-bound receptors to transmit signals into the cell.
intracellular receptor signalling
activation of receptors located inside the cell, typically in the cytoplasm or nucleus, rather than on the plasma membrane. These receptors interact with small, hydrophobic signaling molecules that can cross the lipid bilayer
How do some RTKs activate RAS
A signaling molecule (e.g., Epidermal Growth Factor - EGF) binds to the RTK.
This causes the receptor to dimerize (pair up) and undergo autophosphorylation (adding phosphate groups to tyrosine residues).
**Recruitment of Adaptor Proteins
**
The phosphorylated RTKs serve as docking sites for intracellular signaling proteins.
It then recruits SOS (Son of Sevenless), a guanine nucleotide exchange factor (GEF).
SOS stimulates Ras by exchanging GDP (inactive form) for GTP (active form).
This activates Ras, which then triggers the signaling cascade.
Nerve growth redirection via Rho
Activated RTKs can regulate Rho GTPases through GEF and GAP. These regulators control the balance between active (GTP-bound) and inactive (GDP-bound) Rho proteins.
high Rho activation → Stabilizes stress fibers, leading to axon retraction and guidance changes
