ALtklausur Deck Flashcards
1
Q
Membrane structure - Fluid mosaic model
chiantia
A
Phospholipids – phosphate and fatty acid tails
–-> Hydrophilic phosphates
–-> Hydrophobic fatty acid tails
Two layers
–-> With tails together
Protein
–-> Embedded throughout - integral
–-> Some just surface – peripheral
–-> Motility
2
Q
Structure of a building block of a cell membrane
chiantia
A
3
Q
Stucture of Glycerophospholipids in particular Plasmalogen and Phosphatide
chiantia
A
- Glycerol backbone
- 2 fatty acids usually 1 staurated and 1 unsaturated
- C-3 carbon has phosphoric acid group
2 Types of Glycerophospholipids
1. Plasmogen
2. Phosphatide
–> Phosphatidylethanolamines
–> Phosphatidyserines
–> Phosphatidylcholines
–> Phosphatidylinositol
–> Diphosphatidylglycerol (cardiolipin)
4
Q
What is the diffrence bewteen glycerophospholipids and Sphingolipids?
chiantia
A
- glycerolipids: glycerol backbone, 2 fatty acids, C(3) with phosphoric acid group
- sphingolipids:
sphingosine backbone, 1 fatty acid, sugar –> glycosphingolipid, sphingosine backbone, fatty acidphosphate + alcohol –> sphingomyelin
5
Q
Influenza Virus Membrane
chiantia
A
- assembly, budding in lipid raft domains on apical membrane of infected cells
- hemagglutinin (HA), neuraminidase (NA) are part of lipid rafts→coalescence, enlargement of raft domains
- HA, NA clustering→membrane deformation, initiate virus budding, alter membrane curvature
- M1
–> bind cytoplasmic HA-,NA-tails → M1 polymerizes → interior virion structure
–> docking site for viral RNPs
–> mediate M2 recruitment - M2
–> in cholesterol-rich environment: stabilizes budding site
–> budding virion neck: alters membrane curvature through insertion of amphiphatic helix→ membrane scission, virion release
6
Q
Driving force for domain formation
chiantia
A
diffusional order:
decreased lateral movement
structural order:
same side chains;
unstructural order:
cis side chains
condition:
1.) Gel phase
* T<Tm
* cis→trans isomerization→strong VdW (high structural + diffusional order)→stable system
* packed→decreased lateral movement
* thicker, stiffer membrane
2.) liquid disordered
* T>Tm
* high fluid
* irregular packing
* trans
* cis→reduced accessible surface area to other fatty acid chains→weakend VdW
3.) liquid ordered
* high, rigid sterole→tighter packing + separating gel phase lipids
* all-trans (energy min. configuration)
* rapid lateral diffusion
7
Q
What can you say about the melting Temperature of a saturated fatty acid? Which powers play a role
A
saturated fatty acids have more hydrophobic interactions than unsaturated fatty acids
=> higher melting temp.
8
Q
Which type of membrane lipids preferentially accumulate in lipid rafts?
A
Sphingolipids (e.c. Sphingomyelin) und Cholesterol, Glycosphingolipids
=> higher order and tighter packing of lipids
9
Q
What resourcing power, carbon source and energy source do oxygenic and anoxygenic photosynthesis use?
Dittmann
A
anoxygenic
* energy source: ADP ATP
* carbon source: CO2 (CH2O)n
* resourcing power: H2S S0 SO42-
oxygenic
* energy source: ADP ATP
* carbon source: CO2 (CH2O)n
* resourcing power: H2O ½ O2
10
Q
What are the 2 core proteins of PS1 of oxygenic organisms?
Dittmann
A
The two core proteins of Photosystem I (PSI) in oxygenic organisms are PsaA and PsaB.
* integral membrane proteins that form the core of the PSI complex involved in the light-dependent reactions of photosynthesis.
* capture light energy and transfer electrons during the process of photosynthetic electron transport.
11
Q
Explain the activation of RubisCO
Dittmann
A
- CO2 condenses with RubisCO Lys side chain carbamate
- Carbamate stabilization via Mg2+ (Mg2+ = metal ion cofactor for RubisCO)
- Formation of the carbamate is facilitated by the enzyme Rubisco activase in plants (but can also form spontanously)
12
Q
What is the principle photoreceptor in oxygenic photosynthesis and anoxygenic photosynthesis? To which compound class are these molecules belonging?
Dittmann
A
- oxygenic: chlorophyll
- anoxygenic: bacteriochlorophyll
photosynthetic pigments; substituted circular tetrapyrroles with central Mg2+ atom
13
Q
Describe the principle of the transketolase reaction!
Dittmann
A
transketolase
* requires cofactor thiamin pyrophosphate (TPP)
* transfers C2 from ketose aldose
* catalyzes reactions of calvin cycle
1st reaction:
Fructose-6-P + GAP –> Erythrose-4-phosphate + Xylulose-5-phosphate
2nd reaction:
Seduheptulose-7-phosphate + GAP –> Ribose-5-phosphate + Xylulose-5-phosphate
14
Q
Stochiometry of calvin benson cycle (How much ATP and NADPH)
Dittmann
A
- Fixation of CO2 by RubisCO to form 2 molecules of 3-phophoglycerte (3PGA)
- Reduction of 3 PGA to form hexose sugars
- Regenertion of ribulose 1,5-bisphophate
6 Rounds of calvin cycle are requiered to generate 1 hexose utilizing 18 molecules ATP and 12 molecules NADPH
6 CO2 + 18 ATP + 12 NADPH + 12 H2O –> C6H12O6 + 18 ADP + 18 Pi + 12 NADP+ + 6 H+
15
Q
What is Gout? What can you see in Gout?
Arndt
A
- Purine disease
- Excess and accumulation of Uric acid
- Painful joints (often in toes) due to deposits of sodium urate crystals -> Inflammation
- Primarily affects males
- May involve genetic under-excretion of urate and/or may involve overconsumption of fructose
Treatment: avoidance of purine-rich food
(seafood, liver)
Also treated with xanthine oxidase inhibitor
allopurinol (suicide inhibitor) =>(Hypo)Xanthine excreted
16
Q
Why is arginine-glutamate injected to help against toxic Hyperammonemia?
Arndt
A
Arginine and glutamate injections help in hyperammonemia by stimulating the urea cycle, enhancing ammonia detoxification, and reducing neurological symptoms.
They facilitate ammonia conversion to urea and bind to ammonia directly, reducing its toxic effects
17
Q
Ribonucleotide reductase (RNR) is a target for several anticancer drugs such as gemcitabine. Please explain the consequences for the cell when inhibiting ribonucleotide reductase
Arndt
A
- Deoxyribonucleotides are synthesized from ribonucleoside diphosphates. (e.g. ADP –> dADP)
- 2’-hydroxyl group of ribose is directly reduced to 2’-H bond… without activating the carbon!
- Mechanism: Two H atoms are donated by NADPH and carried by proteins thioredoxin or glutaredoxin
Inhibiting RNR consequence:
can´t form dATP –> no nucleotides for DNA-synthesis –> no proliferation
18
Q
Some anticancer drugs such as methotrexate block dihydrofolate reductase. Please explain how this affects the cells
Arndt
A
- reduces dihydrofolic acid –> tetrahydrofolic acid
- NADPH = e- donor
- necessary for thymine synthesis
- inhibition limits cell growth, proliferation
19
Q
Anticancer drug 5-Fluoro-Uracil is activated by the salvage pathway
Arndt
A
- 5-FU is converted into fluorodeoxy-uridylate (F-dUMP) by cancer cells.
- F-dUMP is a suicide inhibitor of thymidylate synthase (TS).
—> Inhibition of DNA and RNA synthesis
—> 5-FU acts on dividing and non dividing cells
20
Q
Cori and Glucose-Alanine-Cycle
Muscles use mainly ATP from Glycolysis as energy source. Which amino acid is produced as a result and what is it converted into in the liver?
Arndt
A
working muscles operate anaerobically and rely on glycolysis for energy
CORI-Cycle
* Glycolysis produces pyruvat –> lactate (regeneration of NAD+ for glycolysis)
* Lactate is transported to the liver, converted to glucose and transported back to the muscle (for energy)
Glucose-Alanine-Cycle
* imortant to eliminate waste nitrogen from amino acid catabolism and replenish energy as glucose
* Pyruvate –> alanine (transported to the liver) –> Glucose (transported back to muscle)
* Alanine is transported into the liver
* Alanine is then converted into glutamate –> alpha-ketoglutarate
21
Q
What is the Lambert-Beer law?
Bier
A
- exponential decay of light intensity with increasing layer thickness (cuvette)
- number of absorbing photons/time unit (= decay of light intensity dI) on path dL in cuvette is proportional to
–> number of incident photons/time unit
(= light intensity I)
–> number of absorbable molecules
(= product from c and path length dL)
extinction, absorption, absorbance, transmission:
* absorption designates (bezeichnet) a molecular process
* extinction resp. absorbance: observed decay of light intensity: E = log10
* light scattering can also contribute to extinction
* transmission describes medium permeability for waves: T = I/I0
extinction coefficient:
* describes how much radiation is absorbed by substance at 1 cm path length in medium
* dependent on: path length in media (cuvette thickness), c
22
Q
Name the order in which the following molecules are synthesized in the citrate-cycle.
From citrate to Oxalacetate
Wendler
A
23
Q
What is the initial reaction of the citric acid cycle? Name the Substrates and Product.
Wendler
A
condensation of acetyl-CoA with oxaloacetate to form citrate. This reaction is catalyzed by the enzyme citrate synthase.
Substrates:
Acetyl-CoA
Oxaloacetate
Product:
Citrate
24
Q
which steps of the Glycolysis are exergonic and non-reversible
Wendler
A
Phosphoenolpyruvate → Pyruvate.
25
Pair the base according to its function
## Footnote
Wendler
1) Hydrophilic (Threonine)
2) Can act as proton donor and acceptor
at neutral pH (pH=7) (Histidine)
3) Absorbs UV-light (Tyrosine)
4) Is situated in the protein core (Valin)
26
Name the two classes of transposons and the autonomies they can have.
## Footnote
Soultoukis
* TE classification by mechansim
--> replicative vs. conservative
**Class I TE = Retrotransposons**
--> characterised by “copy and paste” mechanism
--> such DNA duplications or transpositions can result in gene duplication, which plays an important role in genomic evolution
**Class II TE = DNA transposons**
--> utilising a “cut and paste” mechanism
27
Explain Histones and nucleosomes
| Soultoukis
* The nucleosome is the subunit of all chromatin
* A single nucleosome consists of about 160 bp of DNA sequence wrapped around a core of histone proteins
* Nucleosome arrays across DNA domains and regions are arragned as „beads on a string“
--> Arrays are folded to form chromatin fibers
* Chromatin fibers are compacted and condensed to form a chromosome
* A nucleosome consists of a segment of DNA wound around eight histone proteins (octamer)
* The histone octamer consist of two copies each of the histone proteins H2A, H2B, H3, and H4
* Each human cell contains around 30 million nucleosomes, each carrying its own epigenetic signature
* Nucleosome positions in the genome are not random, and determine accessibility of DNA to regulatory proteins
* A polynucleosome is a chain of mononucleosomes linked by DNA and histone proteins
28
Explain histone acetylation
## Footnote
Soultoukis
* Newly synthesized histones are acetylated at specific sites
--> Deacetylation occurs after incorporation into nucleosomes
* Acetylation is associated with activation of gene expression
* Acetylation can occur locally or globally (e.g. on sex chromosomes)
--> HATs: Histone acetyltransferases
--> HDACs: Histone deacetylases
--> Group A HATs: act on chromatin
--> Group B HATs: act on newly synthesized histones (cytosolic)
29
Name 3 factors that regulate chromatin structure
## Footnote
Soultoukis
1. Histone modifications
2. Chromatin remodeling complexes
3. DNA methylation
30
Leucine zipper motifs
## Footnote
Soultoukis
* Amphipathic alpha helix
* Leucine in every 7th position
* Allows protein dimerization via interactions between hydrophobic surfaces of two Leucine zipper proteins
* Adjacent positively charged basic region makes DNA contact (bZIP)
31
Recombination
## Footnote
Lenhard
**Homologous recombination** is essential in meiosis for generating diversity and for chromosome segregation, and in mitosis to repair DNA damage and stalled replication forks.
**Site-specific recombination** involves specific DNA
sequences.
**Somatic recombination** – Recombination that occurs in nongerm cells (i.e., it does not occur during meiosis); most commonly used to refer to recombination in the immune system.
--> Recombination systems have been adapted for experimental use.
**Picture 2**
* heterozygous individual that inherited A, B on one chromosome and a, b on the other parental chromosome + no homologous recombination
→ no crossing over → gametes: AB, ab
* homologous recombination between non-sister chromatids→recombinant gametes
* every site that carries sequence homology is potentially a substrate for this recombination activity
* site-specific recombination is driven by sequence-specific recombinase enzymes
* somatic recombination refers to VDJ segments in immune systems during maturation of B/T cells
32
What is the difference between homologous and specialized recombination? Name a biotechnical technique that utilizes specialized recombination.
## Footnote
Lenhard
* Homologous recombination occurs in germ cells initiated by a DSB either DSBR/SDSA; random (depends on the location where DSB occurs)
* Specialized recombination: Cre/lox, Flp/Frt; targeted recombination through recombination target sites (loxP/Frt)
33
DNA Polymerase
## Footnote
Lenhard
**DNA Polymerases are the Enzymes that make DNA**
* DNA synthesis in semiconservative replication + DNA repair reactions
* DNA Pol I: 5 ́-3 ́exonuclease activity that can be combined with DNA synthesis→nick translation
--> nick = ssDNA break in dsDNA
--> nick 3 ́-OH group = initiation site for DNA synthesis o old strand degraded by 5 ́-3 ́exonuclease
* DNA Pol: 3 ́-5 ́exonuclease activity (“proofreading”)→excise incorrectly paired bases
--> E adds base to growing strand
--> wrong base inserted
→ proofreading
→ base hydrolyzed + expelled
→ reconstitute 3 ́-OH group
→new nucleotide inserted
* replication fidelity improved by proofreading by factor 100
34
DNA Polymerase
Holoenzyme Consists of
Subcomplexes
## Footnote
Lenhard
* clamp loader: places clamp (2 β-SU), which provides processivity to E, on DNA
→ encircles dsDNA; transfer process requires ATP hydrolysis
* +coreE(α,β,θ)
* + τ + 2nd core E
→ symmetric dimer
* at least one catalytic core associated with each template strand
→ leading/lagging strand synthesis
* processivity:
E ability to perform multiple catalytic cylces with single template instead of dissociation after each cycle
35
The Clamp controls association of core E with DNA:
| Lenhard
**helicase DnaB:**
interacts with primase DnaG→initiate each Okazaki fragment
**Helicase:**
* unwinds DNA; connected to DNA Pol holoenzyme
* Primase synthesizes short complementary RNA sequence
→ primase dissociates; 3 ́-OH group of RNA sequence provides priming
site
* DNA Pol III extends RNA primer→Okazaki fragment
* large ss-loop extruded→primase interacts with helicase + generates new RNA primer→new Okazaki fragment synthesis
* DNA Pol I: nick translation (nicks between Okazaki fragments)→replace RNA primer with DNA
* Ligase: seals nicks
--> E + ATP/NAD→adduct: E-AMP-complex
--> transferred to free 5 ́-P at nick site
--> cleaved diphosphate-bond
→ 5 ́-P of nick site forms phosphodiester bond between 3 ́-OH and 5 ́-P of nick o
→ ligation reactions in molecular biology
36
What is nonsense-mediated mRNA decay (NMD)? Briefly explain how it works.
## Footnote
Lenhard
* Transcription error/DNA mutations --> premature stop codons --> ribosome disassembles at/after stop codon --> EJC not displaced --> recruits Upf-factor --> recruits decapping enzymes (DCP) --> decap mRNA --> degradation
* = mRNA degradation due to premature stop codon prior to last exon
37
What is the difference between homologous and specialized recombination? Name a biotechnical technique that utilizes specialized recombination.
## Footnote
Lenhard
* both are two distinct types of DNA recombination mechanisms.
**Homologous recombination**
* occurs between two DNA molecules that have significant regions of homology
* DNA strands of the two molecules are broken and exchanged to produce new hybrid molecules that can contain new combinations of genetic material.
* natural process that occurs during meiosis
* responsible for generating genetic diversity in sexually reproducing organisms.
--> It can also be used as a tool in molecular biology to generate targeted mutations, knockouts, and gene replacements.
**site-specific (or specialized) recombination**
* occurs between two DNA sequences that share a specific recognition site or sequence, which is recognized by a specific enzyme or protein.
* This enzyme or protein catalyzes the exchange of DNA strands between the two sites, resulting in the insertion, deletion, or inversion of DNA sequences.
--> Site-specific recombination is often used in biotechnology and genetic engineering to insert or remove specific DNA sequences at precise locations in the genome.
**Example of a biotechnical technique**
* site-specific recombination is the Cre-loxP system
38
Name the key steps in the double-strand break repair model for homologous recombination
## Footnote
Lenhard
* **0th:** DSB (e.g. from ionizing radiation/other damaging processes) introduced on chromosomes in meiotic cell
* **1st:** 5 ́ ends of cut are shortened→3 ́ overhanging ends (bound by SSB)
* **2nd:** invasion of 3 ́ overhanging end into duplex of non-sister chromatid
→bp of 3 ́ end + intact non-sister
chromatid
→3 ́-OH group →DNA-Pol start →DNA-synthesis →strand extension →loop of ss-DNA of intact non-sister chromatid is extruded/displaced (→D loop)
* **3rd:** D loop captures 2nd (top) 3 ́ overhanging end → DNA-synthesis + ligation → double holiday junction
* **4th:** resolved double holiday junctions
* no cross-over end product: starts, ends with same color
* cross-over end product: start, ends with different color
* **heteroduplex DNA:**
contains blue ss of top chromosome + red ss from red chromosome; can contain bp which don ́t fit together
39
Initiation in Bacteria Needs 30S
Subunits and Accessory Factors
## Footnote
Bäurle
**Initiation involves base pairing between mRNA – rRNA:**
* initiation site on bacterial mRNA: AUG initiation codon preceded by Shine-Dalgarno polypurine hexamer (AGGAGG; ~ 10 bp upstream; bacterial mRNA)
* 16S rRNA of 30S bacterial ribosomal SU with complementary Shine-Dalgarno sequence (3 ́)
→base pairing
**Translation initiation in bacteria needs 30S SU and accessory factors:**
* ribosome-binding site
--> sequence on bacterial mRNA
--> includes initiation codon bound by 30S SU in initiation phase in initiation phase of polypeptide translation
* requires separate 30S, 50S ribosome SU + initiation factors (IF-1/2/3), which bind 30S SU
* 30S SU carrying initiation factors binds to mRNA initiation site
→initiation complex
* IF-3 released
→50S SU joins 30S-mRNA complex
40
How do histone posttranslational modifications affect
## Footnote
Bäurle
41
Name four histone modifications
## Footnote
Bäurle
1.Methylation
2. Acetylation
3. Phosphorylation
4. Ubiquitination
42
What is the function of the Shine-Dalgarno sequence in translation? Which organisms have a Shine-Dalgarno sequence?
## Footnote
Bäurle
* Bacteria
* Translation initiation
* initiation site on bacterial mRNA: AUG initiation codon preceded by Shine-Dalgarno polypurine hexamer (AGGAGG; ~ 10 bp upstream; bacterial mRNA)
* 16S rRNA of 30S bacterial ribosomal SU with complementary Shine-Dalgarno sequence (3´) base pairing
43
Mechanisms of Epigenetic inheritance: DNA Methylation
## Footnote
Bäurle
* DNA methylation→ inactivate gene
* replication → hemimethylated state
* DNA methylase methylates other Cytosine
44
Mechanisms of Epigenetic inheritance: Prions
## Footnote
Bäurle
**prion** = a proteinaceous infectious agent that behaves as an inheritable trait, although it contains no nucleic acid.
Examples are:
– PrPSc, the agent of scrapie in sheep and bovine
spongiform encephalopathy, and
– PSI, which confers an inherited state in yeast
Yeast prions show unusual inheritance:
* Sup35
--> in WT soluble form = translation termination factor
--> also as alternative form of oligomeric aggregates, in which it ́s not active in protein synthesis
* spontaneous transition with low frequency
* presence of oligomeric form causes newly synthesized protein to acquire inactive structure
* [PSI+] state forms amyloid fibres
* Purified protein can convert the [psi–] state
of yeast to [PSI+]
45
What are insulator elements?
## Footnote
Bäurle
* Specialised chromatin structures with hypersensitive sites
* Boundary regions between TADs
* Block inhibiting/activating effects of enhancer/silencer
* Block heterochromatin spread
46
How does the genome defend itself against transposons?
## Footnote
Bäurle
* RNAi
* DNA methylation
47
RNAi - How does it work?
## Footnote
Bäurle
**RNAi:**
gene expression silencing due to action of short RNA molecules
**How does RNAi work?**
1. required: long dsRNA
2. dsRNA processed by DICER → siRNA (20-25 nt)
3. siRNA associate with ARGONAUTE effector complex (RISC)
4. complementary mRNA (sequence specificity!) bound by RISC and inhibited
**Where does dsRNA originate?**
* Transgene (hairpin constructs)
* Complex transposon integration
* natural cis-antisense transcripts
* RNA-dependent RNA Polymerase (RDR)
48
Most siRNAs are produced from transposons and repetitive DNA:
## Footnote
Bäurle
* most cellular siRNAs derived from transposons, other repetitive sequences
* in Arabidopsis (seen in picture): high repeat density in pericentromeric chromosome regions
49
Small RNAs, siRNAs and miRNAs
## Footnote
Bäurle
* Small RNAs contribute to the regulation and defense of the genome, and confer silencing specificity through base-pairing
* siRNA targets include repetitive-rich heterochromatin, transposons, viruses or other pathogens
* miRNAs and tasiRNAs targets include regulatory
genes affecting developmental timing or patterning, nutrient homeostasis and stress responses
50
Molecular motors:
## Footnote
Gräf
* dynein: MT; (+) → (-)
* kinesin: MT; (-) → (+)
* myosin: actin; (-)→(+)
Microtubuli end (+) at centromere and (-) at centrosomes
51
Nuclear import and export: the nuclear pore complex (NPC)
## Footnote
Gräf
* transport into nucleus during interphase
* 3000-5000 NPCs/cell
* 90-120 mio Da
* ca. 30 different proteins = nucleoporins = NUPs (orruring in a multiple of 8)
* 16x larger than ribosome
* Diameter: 120 nm
* Pore size: 30-40 nm
* Passive metabolite diffusion; globular protein till 60 kDa (also depends on shape, protein species)
* Disassembly into soluble subcomplexes during mitosis; re-assembly starts in telophase
* up to 1000 molecules/s
* export: RNPs, ribosome SU, t/mRNAs
* import: TFs, chromatin components, ribosome proteins, nuclear lamina components
* transport requires NLS/NES; retention: NRS
52
Specific nuclear export:
## Footnote
Gräf
Specific nuclear export:
* export receptor binds NES + Ran-GTP = export complex
* interaction: export complex + FG-repeats → shuttle
* Ran-GDP dissociates export complexes
53
Specific nuclear import and export
## Footnote
Gräf
FG repeats:
* In NPC
* natively unfolded → no structure; mesh of protein chains → barrier for proteins > 60 kDa → bigger proteins have to phase separate into mesh
* other solubility environment
→ “hydrogel” structure with H2O
* →phase separation
* NTF-binding sites
54
Nuclear mRNA processing
5’ mRNA capping
## Footnote
Soultoukis
* The 5'-ends of all eukaryotic pre-mRNAs studied so far are converted to cap structures
* The cap provides structural stability and various functionalities to the mRNA molecule
* Cap is required for mRNA export to the cytoplasm
* Caps influence translation initiation and splicing of the first intron
* The cap is bound by 'cap-binding' proteins, and provides resistance to mRNA exonuclease degradation
* The capping reaction usually occurs very rapidly on nascent transcripts; after the synthesis of only a few nucleotides by RNA polymerase II.
55
What is the difference between open and closed mitosis?
## Footnote
Gräf
open mitosis: the segregation of chromosomes takes place after the nuclear envelope breaks down
closed mitosis: the segregation of chromosomes takes place without the nuclear envelope breaking down.
56
You use synchronized cells in G2 phase and inhibit the polo kinase (Plk1) – at which point do you thereby stop the cell cycle and the activity of which protein thereby fails immediately which is responsible for the stop of the cell cycle
## Footnote
Gräf
* Inhibiting Polo-like kinase 1 (Plk1) halts the progression of the cell cycle.
* Plk1 plays a crucial role in regulating mitotic entry, progression, and exit.
* Plk1 is particularly involved in the transition from G2 phase to mitosis (M phase).
* Cells arrested at the G2/M checkpoint upon Plk1 inhibition cannot enter mitosis.
* Plk1 activity is immediately required for proper cell cycle progression, especially during the G2 to M transition.
57
What do centrosomes and cilia have in common?
## Footnote
Gräf
* Both centrosomes and cilia are microtubule-based structures found in eukaryotic cells.
* Both play essential roles in cell division, organization, and cellular motility.
* They are involved in various cellular processes, including cell division, intracellular transport, and signal transduction.
* Centrosomes serve as the main microtubule-organizing centers (MTOCs) in animal cells, while cilia often arise from basal bodies derived from centrosomes.
* Centrosomes contribute to spindle formation during mitosis and meiosis, while cilia are involved in cell motility and sensing extracellular signals.
58
In which cell cycle phase is the centriole formed?
## Footnote
Gräf
during the interphase
In this phase, the cell undergoes DNA replication, and centrioles replicate as well. This ensures that each daughter cell will receive the necessary complement of centrioles during cell division.
59
The Casparian strip forms a diffusion barrier in the cell walls of root endodermal cells:
## Footnote
Grebe
* Casparian strip (apoplastic → symplastic transport)
--> discovered by Robert Caspary
--> Lignin → H2O-impermeable → pass through plasma membrane + cytoplasma of endodermal cells
→control transport: H2O; inorganic salts between cortex, vascular bundle o ring-like
--> root endodermis of vascular plants
* Casparian Strip Domain (CSD) = Plasma-membrane domain of
differentiated endodermal cells in direct contact with C
60
Differentiation of Casparian Strip in the root of A. thaliana:
## Footnote
Grebe
* root hair + casparian strip in differentiation zone (not-growing)
* separates cortex apoplast – vascular tissue apoplast
61
The Casparian strip in the root endodermis of A. thaliana contains lignin:
## Footnote
Grebe
* PA: can ́t diffuse into vascular system through Casparain strip
* No lignin→no casparian strip made; molecules diffuse into vascular system
* PA + monolignols→Casparian strip made→no diffusion
62
Inactivation of BIN2 repressor kinase:
## Footnote
Grebe
* found in cabbage, cauliflower (first isolated from pollen)
* BR-binding to BRI1 → BRI-BAK1 heterodimers + phosphorylation
* BR-signaling kinase (BSK) is phosphorylated
* BSK activates BSU1 (phosphatase) → BIN2 dephosphorylation → degradation → BES1, BZR1 not longer phosphorylated → active
* BR absence → BIN2 active (nucleus) → BES1, BZR1 phosphorylation → degradation
63
Pectins
## Footnote
Grebe
--> Group of Carbohydrates in cell wall
* abundant in middle lamella, primary wall
* protopectin is mixture of: galacturonans + rhamnogalacturonans
64
Hemicelluloses:
## Footnote
grebe
* Location: primary wall
* pentoses (e.g. D-xylose, L-arabinose) + hexoses (e.g. D-/glucose/galactase/mannose)
* golgi: pectin synthesis → vesicle transport via actin to tip
Example: Xyloglucan
--> Picture
Cellulose: only beta-1,4-glucan chain
65
Cellulose:
## Footnote
grebe
* in primary (10%), secondary wall (94%)
* linear beta-1,4 glucan; beta-D-glucose (or its dimer)
* up to 15,000 glucose SU; straightened out
* synthesized by cellulose synthase (in cell wall Cellulose-Synthase complexes)
66
The endoplasmic reticulum (ER): gateway of the secretory pathway
## Footnote
Sauer
* all eukaryotic cells
* intracellular compartment: lipid bilayer membrane enclosing an inner space (lumen)
* = network (“reticulum”) of interconnected membrane tubules, cisternae stretching across entire cytoplasm (“endoplasmic”)
* **synthesis location of: membrane proteins, soluble cargo proteins, lipids → transported to other compartments/extracellular space along secretory pathway**
67
Golgi apparatus:
## Footnote
Sauer
* synthesis of cell wall components (pectins, hemicellulose); cellulose synthesized at plasma membrane
* protein glycosylation continues (start in ER)
68
Which complex is involved in the degradation of membrane proteins in plants?
Briefly explain how it works.
* complex: Multivesicular bodies (MVB)
* membrane proteins are internalized via clathrin-mediated endocytosis (= early endosomes)
* EE fuse together --> MVB
* MVB fuses with vacuole --> degradation
69
How to degrade transmembrane proteins? – MVB/Prevacuolar Compartment
## Footnote
Sauer
Late endosomes (LE)/Multivesicular Bodies (MVB) contain intraluminal vesicles (ILVs)
**1. endocytosis:**
* ligand binds mytogene (EGF)
* dimerization
* switching-off signaling by receptor-mediated endocytosis via clathrin-coated vesicles
**2. gathering:**
* CCV-transport to endosomes
* interaction: clathrin - cytosolic receptors
**3. concentration:**
* receptor concentration at the membrane
* Ub-labeling interaction of receptors with ESCRT complex
* ESCRT complex assembly by PIP3
**4. budding inward:**
* bulge formation in the endosome (away from cytoplasm)
* membrane fusion
* vesicle formation in endosome
* = MVB
**5. fusion MVB lysosomes:**
* hydrolases released into endosome→degradation (vesicles with EGF receptor)
70
Autophagy – Degrading protein aggregates and organelles:
## Footnote
Sauer
71
Steps of Autophagy
## Footnote
Sauer
= process by which cell break down and utilize their own components
**Phases**
* Initiation: three At9-vesicles
* Phagophore (double-membrane) nucleation
* Elongation and autophagosome formation: membrane-addition +
cargo internalization; fusion of phagocytic cup opening →
autophagosome
* autophagosome-vacuole (lysosome in mammalian cells) fusion o Cargo degradation: inner membrane + content are hydrolyzed
* autophagosomes take up cellular material (e.g. misfolded proteins/whole organelles to be degraded); fuse with lysosomes→autophagolysosomes
72
Exocytosis process:
## Footnote
Sauer
Exocytosis (= secretion) needs v(esicle)-SNAREs and t(arget)-SNAREs for docking, fusion:
**SNAREs**
* form highly stable protein-protein interactions
→help to overcome energy barrier required for membrane fusion
* 57 different types in Arabidopsis
▪ belong to different classes
▪ each class has different members
▪ each member has a specific function in a specific transport route
## Footnote
* vSNARE binds t-SNARE * →vesicle pulled to
membrane
73
Explain the V-type ATPase
## Footnote
Baumann
* ~ 900 kDa
* 14 different subunits / in total ~ 30 subunits
* V1 domain: ~ 650 kDa, A3B3CDE3FG3H
* VO domain: ~ 260 kDa, ac8c´c´´de
Function:
* Vacular membranes in plants, yeast other fungi
* Endosomal ans lysosomal membranes in animal cells
* Plasma membrane of osteoclasts and some kidney cells
74
Explain the Sodium Pump (P-Type ATPAse)
## Footnote
Baumann
**Na+/K+-ATPase = Sodium pump**
general structure:
* α subunit of̴ 105 kDa with 10 transmembrane segments
--> ATP-hydrolysis & ion transport
* glycosylated β subunit
--> required for the transport of
newly synthesized pumps to the PM
general function:
* establishment of K+ and Na+ gradients across the PM
**exchanges 3 Na+ for 2 K+
--> electrogenic (inside --> negativ)**
* large conformational changes -->
2 distinct enzymatic states:
E1 & E2
* conformational change triggered by autophosphorylation & dephosphorylation on a conserved Asp residue
Picture:
blue: K+
yellow: Na+
75
Explain ABC Transporters: Floppase
## Footnote
Baumann
**Floppase: (ABC Transporter) moves phospholipids from cytosolic to outer leaflet**
*Flippase: (P-type ATPase) moves PE and PS from outer to cytosolic leaflet*
*Scramblase moves lipids in either direction, toward equilibrium*
* ATP binding at NBD / NBD interface
* substate binding site faces either outward or inward
* ATP binding --> outward-facing conformation
* ATP hydrolysis --> inward-facing conformation
76
The Nernst Equation
## Footnote
Baumann
R = gas constant (8,3144 J* mol-1K-1)
T = absolute Temperature (Kelvin)
z = ion charge
F = Farady constant 96485,3365 J* V-1mol-1
77
What is the name of the response pattern depicted here?
## Footnote
Baumann
* oscillating, frequency-modulated
78
Ion Inside & Outside of a Mammalian Cell
## Footnote
Bauman
Note: [negative charges]cytosol ≈ [positive charges]cytosol
**Conclusion:**
there are huge concentration gradients for various ions across the plasma membrane
79
Patch Clamp:
## Footnote
Baumann
**Action potential propagation**
* open Na channel → Na influx → membrane depolarization → AP
* membrane depolarization propagation passive and with attenuation in both directions o refractory Na channels → no new AP
* unopened Na channels → AP
* →one direction of conduction (decreasing amplitude (with decrement, local response))
Picture 1:
→ study ionic currents inindividual isolated living cells, tissuesections, or patches of cell membrane
* Whole-cell recording: micropipette in tight contact with cell membrane → prevents current leakage; voltage applied → forming: voltage clamp → membrane current measured
* Inside-out recording: attach cell membrane to tube → exposing its cytosolic surface
* This gives access to the surface through the electrolyte solution bath. This method is used when changes
are being made at the intracellular surface of the ion channels.
* Outside-out recording: membrane ruptured → electrode out of cell → original outside is now on the inside
→ enabling studies of the inner membrane surface
Picture2:
* Voltage control across membrane
* → record coloumns
* channels open at depolarisation beginning
* K: channels open delayed
Picture 3:
* Apply voltage → measure current (linear relation; → channel open all the time; voltage-independent)
* Top right: below voltage no current (closed), above (open)
* Bottom right: open channel at neg. voltages, closed at
depolarisation?
80
G-Protein-coupled receptors (GPCRs)
## Footnote
Baumann
* largest family of membrane-bound receptors: 1000-2000 (> 1% of human genome)
* evolutionary quite old
* detect various extracellular signals
* one signal may be different GPCRs → differential expression, reactions
* > 50% of pharmaceutica act on GPCRs and/or GPRC-dependent signaling
**Structure**
* 7 transmembrane α-helices (“7-pass transmembrane receptors”/”heptahelical receptors”)
* N-terminus (E1): extracellular
* C-terminus (C4): cytosolic
* Signal binding: either via transmembrane domains, E1, extracellular loops (E2/3)
* → binding, activation of heterotrimeric G-proteins via C3/4
* Often: palmitoyliaction of Cys in C-terminal region
GPCR act via heterodimeric G Protein:
* 3 SU: αβγ
* Membrane-attached via fatty acyl chains on α, γ SU
* α SU: GDP-bound; GPCR interaction; GTPase
* activated GPCR = GEF for α SU
* target protein = GAP → GTPase of α SU high
81
Cells react to signals from the exterior
## Footnote
Baumann
* exposure to something → cell → response
* cells also have to sense their neighbours → signaling between them (called cell-cell signaling)
**Types of cell-cell signaling**
1. contact-dependent
2. gap junctions
3. autocrine
4. paracrine
5. synaptic
6. endocrine
**autocrine:** cell secretes a hormone or chemical messenger (called the autocrine agent) that binds to autocrine receptors on that same cell
**synaptic:** signal producing cell (neuron); signal at synapse → target cell: postsynaptic cell (neuron/muscle cell); translation: elec. signaling → chem. signaling
**paracrine:** dependent on endocrine cells; hormones released in blood stream; cell produces a signal to induce changes in nearby cells
**hundreds of different signaling molecules:**
* peptides & proteins
* amino acids
* nucleotides
* steroids
* fatty acid derivatives
* gases
82
The heart is an electrical organ. Prior to cardiomyocyte contractions, action potentials result in the release of intracellular Ca2+ from the sarcoplasmatic reticulum. Name those cellular junctions between cardiomyocytes that are involved in the electrical coupling of cardiomyocytes!
## Footnote
Seyfried
Gap junctions
83
What are the three hormones produced within the endocrine glands of the pancreatic Islands of Langerhans?
## Footnote
Seyfried
* Alpha cells: Glucagon
* Beta cells: insulin
* D cells: Somatostatin
84
What are the two characteristics of morphogens?
* Diffuse over several cells (with a decrease in concentration)
* Induce a specific cell fate
85
Select the correct quote on how diffusion from the capillary system is mediated to the interstitium (multiple choice):
a. Colloid osmotic pressure in capillary, hydrostatic pressure in interstitium.
b. Hydrostatic pressure in capillary, colloid osmotic pressure in interstitium.
c. Hydrostatic pressure in capillary, hydrostatic pressure in interstitium.
d. Colloid osmotic pressure in capillary, hydrostatic pressure in capillary.
## Footnote
Seyfreid
b. Hydrostatic pressure in capillary, colloid osmotic pressure in interstitium.
86
Within cardiomyocytes binding of Myosin to Actin filaments is controlled by a regulatory protein complex compromised of several proteins. Name 4 regulatory proteins of this complex
Troponin C
Troponin I
Troponin T
Tropomyosin
* Ca2+-binding to TnC (4 binding sites; all binding sites bound
→ conformational change)
* Troponin-Tropomyosin-complex changes
→ actin myosin-binding sites demasked
* Myosin-ADP-Pi-complex binds actin
→ actomyosin-ATPase activation
* Myosin-ADP-Pi→ADP-Pi release → myosin conformational change → force
TnT = Tropomyosin-binding subunit
TnC = Ca2+-binding subunit
TnI = inhibitory subunit
