11 MZB II - Faresse

- Large protein complex (≈100 proteins), 3000-4000 pores/nucleus
- Bidirectional transport : Active and Passive

Das Kernlokalisierungssignal (auch Kernlokalisationssignal oder Kernlokalisierungssequenz, englisch nuclear localization signal, abgekürzt NLS) ist eine aus wenigen Aminosäuren bestehende Signalsequenz, die Proteine tragen, die in den Zellkern eingeschleust werden sollen

Sequence of 4-8 amino acids positively charged (Arg, Lys, Pro)

- Chromatin = DNA + RNA + Protein (Histone + Non-Histone)
- Histone proteins maintain the 3D conformation of the DNA
- Non-Histone proteins are involve in the replication, reparation and transcription of the DNA

Euchromatin: Active chromatin
Heterochromatin: Inactive chromatin

- Darker zone in the nucleus (1-2 nucleolus / nucleus)
- Site of ribosome synthesis and assembly
- More visible in cells with high synthesis activity (neurons, hepatocytes)

Nucleus disorder
-> can lead to several disorders, one of them is progeria

highly accelerated aging process

- Protein-manufacturing complex composed by ≈80 proteins and 4 ribosomal RNA (rRNA)
- Free in the cytoplasm (synthesis of cytosolic proteins) or attached to membranes of the endoplasmic reticulum (synthesis of membrane or secreted proteins)

- ‘Rough endoplasmic reticulum’ RER
  ‘Smooth endoplasmic reticulum’ SER are separate networks of membrane
- RER and SER represent 50% of the total membrane of the cell

- RER is involved in the production, folding and quality control of proteins
- SER is largely associated with lipid (fat) manufacture, steroid hormone production and detoxification

- The entry of phospholipids in the lipid bilayer is performed in the cytosolic side
- «Flippase» are needed to transfer the phospholipids from the cytosolic layer to the luminal layer

Accumulation of misfolded protein leads to:

- Creutzfeld Jakob
- Alzheimer
- Parkinson
- Huntington

- Succession of flattened disclike structures located close to the endoplasmic reticulum
- Receives proteins and lipids from the RER and SER, modifies, sorts, concentrates and packs them into sealed vesicles

KDEL
- KDEL is an aa sequence : Lys-Asp-Glu-Leu
- Keeps protein in the ER
- Responsible for the retrograde transport of proteins from the Golgi apparatus

- Vesicles filled with hydrolytic enzymes (≈40) degrading material from outside and life-expired components within the cell
- Digestion occurs under acidic condition (pH≈5)
- 2 types of lysosomes: Conventional (ubiquitous) Secretory (T lymphocytes)

s.B.

Worm-shaped organelle with various size involved in energy production

4 different compartments:
- Matrix: Contains proteins and mitochondrial DNA, several enzymatic reactions
- Inner membrane: Folded into several cristae and contains enzymes involved in ATP synthesis
- Outer membrane: Permeable to all molecules < 5000 Da due to numerous channels called “Porin”
- Intermembrane space: Site of H+ accumulation required to produce ATP

-> Closely related to bacteria

• Magnification is the number of times an image's size is enlarged.
• Resolution is a measurement of how well the smallest details of an image can be discerned.

Object that’s less than half the wavelength of the microscope’s illumination source is not visible under that microscope.

Light microscopes use visible light which has a minimum wavelength of 400 nm
-> This means that we will never be able to see any object smaller than approximately 200 nm using a light microscope

The wavelength of electrons is thousand of times shorter than visible light (0.4 nm).
-> This means that we will be able to see objects of approximately 0.2 nm using an EM.

3 elements are required:
- A bright light focused onto the specimen by a condenser.
- A specimen thin enough to allow light to pass through it.
- An appropriate set of lenses to focus the image in our eye.

1. Fixation
- Stop all biochemical reactions
- Preserves tissues from decay
- Generally with Aldehydes PFA or GA
2. Dehydration Embedding
- To prepare embedding
- Baths of alcohol to remove the water
- Bath of Xylol to remove alcohol
3. Embedding
- To harden the tissue
- Immersion in melted, paraffin, resin, or plastic
- Hard after cooling, easier to cut
4. Sectioning
- Using a Microtome
- Light microscope (210 µm)
- Electron microscope (40-100 nm)
5. Staining
- Rehydration using a baths of diluted alcohols
- Immersion in staining solutions

Immunostaining is a general term in biochemistry that applies to any use of an antibody-based method to detect a specific protein in a sample

Fluorochrome, Fluoreszenzfarbstoffe, Moleküle, die in der Lage sind, eingestrahlte Energie in Form von Fluoreszenzlicht (Fluoreszenz) wieder abzugeben
-> Licht mit einer bestimmten Wellenlänge wird Absoriert und anschliessend wird licht mit einer anderen bestimmten Wellenlänge abgegeben

Filter 1: allows only wavelengths that excite the fluorochrome
Filter 2: allows only the light emitted by the fluorochrome

Mit einem confocalen Mirkoskop lässt sich eine viel dünnere Ebene Betrachten als mit einem herkömmichen Mikroskop

The key part of the confocal is the pinhole. It allows only the light from the focal plane to pass through the pinhole to the detector
-> je kleiner dass pinhole, desto dünner die fokusebene (und desto besser die auflösung)

Im Prinzip sehr ähnlich zum Lichtmikroskop

s.B.

s.B.

Prokaryotes:
- Spherical, rod-like or spiral shaped
- Small (few micrometers long)
- Possess a cell wall surrounding the plasma membrane
- One single compartment (cytoplasm + DNA)
Eukaryotes :
- Present a nucleus and organelles
- Bigger that prokaryotes
- More elaborated
- Numerous shapes

s.B.

Lipid bilayer (5 nm) with protein inclusions

- Prokaryotes have a single plasma membrane surrounding the cell
- Eukaryotic cells contain in addition an abundance of internal membranes enclosing intracellular compartments