What are the primary functions of a mitochondrion?
- Generate ATP by cellular respiration
- Regulate cell death - apoptosis
- Synthesize phospholipids - cardiolipin
Explain how mitochondria are dynamic
- move around on microtubules
- undergo membrane fusion and fission
- ensure maintenance of mitochondrial circular DNA
Explain the five stages of mitochondrial protein import.
- 1) Chaperone binds the polypeptide in the cytoplasm and transports it to the mitochondrion.
- 2)A sequence in the polypeptide directs it to the Translocon of the Outer Membrane (=TOM) complex which is a large transmembrane channel that drives the polypeptide through theouter membrane into the intermembrane space.
- 3/4) The small Translocon of the Inner Membrane (=TIM) complex, which is a peripheral membrane binds the polypeptide delivers to the large TIM complex, which is a large transmembrane protein that inserts the polypeptide into the inner mitochondrial membrane membrane.
- 5) Polypeptide is released into the membrane and it folds into its native structure
What kinds of molecules are found in the matrix of mitochondria?
- 50% protein
- mitochondrial ribosomes, tRNA, mRNA
- location of TCA cycle
How many genes are encoded in the mitochondrial cDNA?
Provide an example of the clinical relevance of mitochondria.
- mitochondrial myopathy- caused by impaired mitochondrial structure, which leads to impaired energy production
- specific example is Mitochondrial Encephalopathy Lactic Acidosis and Stroke-like [=MELAS] episodes, which is characterized by aberrant cristae architecture and mutations in the mitochondrial tRNA which, in this case, is encoded by the mitochondrial DNA
- mutations in the nuclear genome or mitochondrial genome can both lead to mitochondrial disorders
How large are mitochondria?
up to 10 um in length and 0.5-1 um in width
How large are peroxisomes?
approx 1 um in diameter
What are the primary functions of peroxisomes?
- regulation of metabolism - beta-oxidation (breakdown of fatty acids)
- contain over 40 enzymes
How are proteins transported to peroxisomes?
- 1. from the cytoplasm via a peroxisomal targetting signal sequence in the protein
- 2. from the ER by fusion with ER-derived vesicles
Provide an example of the clinical relevance of peroxisomes.
Zellweger Syndrome is caused by a defect in peroxisomal protein import- leads to problems in the brain, liver, and kidney. Death occurs soon after birth.
What are the three primary functions of the cytoskeleton?
- 1. Maintain cell morphology
- 2. Move intracellular components
- 3. Facilitate the movement/migration of entire cells
What are the three major classes of cytoskeleton?
- 1. Thin filaments (Actin)
- 2. Intermediate filaments (nuclear lamins)
- 3. Microtubules (Tubulin)
Describe the structure of thin filaments.
- F (fibrous) actin filaments are formed from two chains of G (globular) actin monomers
- Filaments are dynamic with plus (faster growing) ends and minus (slower growing) ends.
- Capping proteins regulate the length and speed of growth.
- Filaments can be bundled together for different functions, and bundles are maintained by different actin binding/stabilizing proteins (eg. α-actinin)
- Myosin motors can attach to F-actin to either initiate contraction (both in muscle and nonmuscle cells) or move cargo (vesicles)
What are focal adhesions?
- Regions where the actin cytoskeleton connects to the ECM
- Allow the cell to exert tensile force on the ECM
- Important for wound healing
- Contractile bundles connect to the ECM via integrins
- Stabilized by vinculin and talin
What role do cortactin and filamen play in the cytoskeleton?
They are network regulators which link actin filaments together into gel-like networks in the outer portion of the cell - critical for maintaining cell shape.
Describe the structure of microtubules.
- hollow tubes comprised of 13 parallel protofilaments of alpha and beta tubulin heterodimers
- originate at the centrosome from gamma tubulin ring complexes
- have a plus end where elongation occurs via the addition of tubulin dimers and a minus end where shrinkage occurs via the removal of dimers (stabilized by gamma tubulin)
Name three functions of microtubules.
- 1. Movement of organelles
- 2. Ciliary and flagellar motion
- 3. Chromosome segregation
Name the two families of motor proteins associated with microtubules.
- 1. Kinesins (move towards plus end)
- 2. Dyneins (move towards minus end)
What are centrioles?
Centrioles are specialized microtubular structures within centrosomes, nucleate microtubules and generate the long spindles that attach to chomosomes during mitosis
Describe the structure of intermediate filaments.
- 8-10 nm in diameter, formed by overlapping protein rods
- Form structural scaffolds within the cytoplasm (eg. cytokeratins) and the nucleus (eg. lamins)
What is the difference between euchromatin and heterochromatin?
- Euchromatin is unwound DNA, appears light on an electron micrograph
- Heterochromatin is wound DNA, appears dark on an electron micrograph (electron dense)
What is a nuclear pore?
- large protein assembly embedded in the nuclear envelope with 9-11 nm pore
- connected to each other by lamins (Intermediate filaments)
- controls entry and exit from the nucleus
What does the number of nuclear pores correlate with?
metabolic activity of the cell
Fill in the blanks and explain each step.
- 1. Capture: In the cytoplasm cargo proteins with a 'Nuclear Localization Signal' (NLS) interact with importin proteins
- 2. Import - protein-importin complex is moved into the nucleus through the nuclear pore complex
- 3. Release - Once in the nucleus RanGTP binds the cargo/importin complex and the cargo is released (allows for the recycling of importins)
- 4. Export - RanGTP associated with importins alpha or beta or any protein with an nuclear export signal (leucines) are exported out of the nucleus into the cytoplasm
- 5. Release - -Once in the cytoplasm the action of 'GTPase activating proteins ('RanGAPs') convert RanGTP to RanGDP. This releases the importins proteins so that they can begin the process again with new cargo proteins.
- 6, Diffusion - RanGDP can passively diffuse into the nucleus through the nuclear pore
- 7. Ran Activation - In the nucleus, Ran Guanine Exchange Factors (RanGEFs) convert RanGDP to RanGTP so that it is once more active and ready to export importins once again
Fill in the blanks.
- 1. Transcription
- 2. Nuclear Import (Importins)
- 3. Release
- 4. Nuclear Export (Ran)
- 5. Translation of mRNA
Name the four stages of the cell cycle.
- G1 (gap) - cell prepares to copy genome
- S (synthetic) - DNA duplication
- G2 - preparations for mitosis (make tubulin)
- M - mitosis
Which combination of cyclins (Cln) and cyclin-dependent kinases (Cdk) are present in the G1 phase?
Which combination of cyclins (Cln) and cyclin-dependent kinases (Cdk) drive the cell from G1 to S phase?
Which combination of cyclins (Cln) and cyclin-dependent kinases (Cdk) drive the cell from S to G2?
Which combination of cyclins (Cln) and cyclin-dependent kinases (Cdk) drive the cell from G2 to mitosis?
How does the cell ensure that each stage was completed correctly?
Explain how the p53 checkpoint works.
DNA damage (ie. mutation) activates p53 which then induces p21 that then inhibits CyclinE/CDK2 such that the cell does not move from G1 to S. This gives the cell a chance to repair the DNA damage/mutation. If it cannot, p53 can then direct it to undergo apoptosis.
programmed cell death
What are the functions of apoptosis?
- reduced inflammation compared to necrosis (cell lysis)
- controls cell proliferation in development
- response to cellular damage by clearing damaged cells
Describe the extrinsic pathway of apoptosis.
- 1. extracellular death ligand binds receptor
- 2. Activated receptor activates caspase 8 (protease)
- 3. Caspase 8 cleaves and activates downstream "executioner" caspases (3, 6, 7)
- 4. Executioner caspases induce membrane blebbing, proteolysis of cytoskeleton, and digestion of chromosomes
Describe the intrinsic pathway of apoptosis.
- 1. UV induces DNA damage
- 2. DNA damage activates p53
- 3. p53 activates BAX and BAK
- 4. BAX and BAK induce cytochrome C release from mitochondria
- 5. cytochrome C activates apoptosome (APAF1)
- 6. apoptosome cleaves and activates caspase 9
- 7. caspase 9 cleaves and activates executioner caspases