Biologie Cellulaire: Le Noyau

• Biology cellular course sheet n°10 focuses on the nucleus.

Nuclear Envelope

• The nuclear envelope has an outer membrane that faces the cytoplasm.
• The nuclear envelope has an inner membrane on the nucleoplasmic side.
• Nuclear pores allow exchanges between the nucleus and the cytoplasm.
• The lamina lines the inner nuclear membrane.

Chromatin and Nucleolus

• Chromatin is an association of DNA with proteins, especially histones.
• The nucleolus is involved in ribosome synthesis.

General Characteristics of the Nucleus

• The nucleus occupies about 10% of the total cell volume on average.
• Lymphocytes have a large nucleus while squamous cells have a very small nucleus.
• The nucleocytoplasmic ratio indicates nuclear size relative to cytoplasm volume.
• The nucleus isolates DNA from the cytoplasm.
• DNA is mainly in the nucleus, with a small amount in mitochondria.
• Unlike eukaryotes, prokaryotes lack a nucleus.
• Eukaryotes have a nucleus, prokaryotes do not.

Roles and Characteristics of the Nuclear Envelope

• The nuclear envelope delimits the nucleoplasm.
• The nucleoplasm is a partially enclosed compartment.
• There are zones of assembly between the external and the internal membranes.
• Nuclear pores allow regulated nucleocytoplasmic traffic.
• The perinuclear space is continuous with the ER lumen.
• Intermediate filaments of the cytoskeleton are involved in nuclear protection.
• A perinuclear basket may be present in cells under stress, such as keratinocytes.
• The nuclear lamina or nucleocortex maintains nuclear shape.
• It is linked to the external membrane via transmembrane proteins.

Roles of the Nucleus

• The cytoskeleton protects DNA from movement, especially microtubules.
• The nucleus protects DNA from cytoskeletal damage.
• In bacteria, transcription and translation occur simultaneously in the cytoplasm.
• In eukaryotes, transcription occurs in the nucleus, and translation in the cytoplasm. This separated sequence allows for alternative splicing,.
• Alternative splicing can create different proteins from the same gene.
• Ribonucleoproteins (RNPs), which are associations of RNA and proteins, are used in splicing.

Nucleoplasm Content

• The nucleoplasm contains chromatin and the nucleolus.
• Chromatin exists in two compaction levels: heterochromatin and euchromatin.
• Heterochromatin is highly compact and dense with electrons.
• Euchromatin is less dense and appears clearer with electrons.
• The nucleolus is a dense area visible with electrons.
• The nucleolus is not delimited by a membrane.

Nuclear Exceptions

• During maturation, cells may lose their nucleus.
• An example of a cell without a nucleus is the erythrocyte, which develops from the erythroblast.
• Erythrocytes are specialized cells lacking a nucleus or organelles.

Multinucleated Cells

• Hepatocytes in the liver are binucleated.
• Physiological examples of multinucleated cells include hepatocytes.
• Giant cells can fuse their plasma membranes, sharing cytoplasm while retaining individual nuclei to form Syncytium.
• Pathological examples include cells infected by HIV.

Nuclear Envelope Membranes

• The external membrane is continuous with the endoplasmic reticulum membrane.
• The external membrane has biochemical similarities with the ER.
• There are ribosomes on the cytosolic side of the external membrane.
• The external membrane has transmembrane proteins, particularly enzymatic ones.
• The internal membrane has different composition than the external membrane.
• The internal membrane lacks enzymatic proteins and ribosomes.
• The internal membrane has abundant transmembrane proteins that serve as receptors for lamins and histones, and is linked to chromatin.

Nuclear Lamina

• The nuclear lamina is a meshwork of proteins that covers the nucleoplasmic side of the internal membrane while not covering the ports.
• Lamins A, B, and C make up the nuclear lamina.
• Lamins A and C are similar due to alternative splicing of the same gene.
• Lamina is part of the intermediate filament family.

Comparing Nuclear Lamina and Intermediate Filaments

• Lamina has bidimensional mesh and filaments have tridimensional mesh.
• Lamina has a longer central domain
• Lamina has a NLS sequence that targets it to the nuclear pore.
• Lamina is dynamic and depends on phosphorylation, whereas filaments are static.

Nuclear Lamina Dynamics

• Phosphorylation of serines in lamin sequences causes loss of affinity and network destabilization during the start of mitosis (prophase).
• The phosphorylation begins during prophase.
• Nuclear envelope fragmentation into small vesicles happens during prophase.
• Some lamins are solubilized, while others remain bound to nuclear envelope fragments with pores.
• Dephosphorylation of lamins in telophase restarts lamina.
• Envelopes form around replicated chromosomes in each daughter cell, and soluble parts are added.
• Proteins excluded from chromosomes must regain entry via nuclear pores.

Laminopathies

• Laminopathies are diseases caused by lamin dysfunction.
• Laminopathies are rare, affecting about 1 in 6 million births.
• Progeria is characterized by rapid aging, is a well-known Laminopathy.
• Progeria involves aging 10x faster, and affected children have a life expectancy of 13 years, mostly affecting fast growing tissues.

Nuclear Pores

• There are 3000-4000 pores per nucleus, covering about 1/3 of the surface area, varying amount depending on protein synthesis levels.
• Each port is made of 100 nucleoporins in octagonal.
• There are three main elements of pores: Column subunits, annular ring subunits, and luminal subunits.

Structure of Nuclear Pores

• Column subunits are positioned around the port.
• Ring subunits are around the edges of the port
• Luminal Subunits are transmembrane proteins, and anchor the port to the nuclear envelope.
• Other proteins are on cytosolic and nucleoplasmic sides.
• Eight ring sub-units are bound on the elements in a column
• Eight fibrillar sub-units: Present on each side on the envelope, form a nuclear cage.
• Some proteins on the side of the pore obstruct the lumen.

Visualizing Nuclear Pores

• Visualization is done by lysing cells in detergent and studying purified pores.
• Proteins in the center of the pore are hard to show do to loss of proteins during purification.

Nucleocytoplasmic Traffic Through Nuclear Pores

• Allow the passage of: Small proteins, nucleotides, and ions.
• Molecules must be soluble in water.
• The molecule weight must be under 40kD.
• 9 nm of free space allows passage of small molecules.
• Small spaces are created between column and annular elements.
• A larger molecule takes longer to pass through the pores than a smaller one.
• Proteins meant for the nucleus use a specific import mechanism.
• Proteins have a small Nuclear Localization Signal (NLS) sequence.
• Import is incredibly fast, regardless of molecular weight.

Protein Import into the Nucleus

• Present anywhere
• 4 to 8 amino acids are positively charged : rich in lysine and arginine.
• Protein cytosol is the sequence of the NLS in the cytosolpasme
• Dirlge protein towards the NLS, and takes in components
• The protein is formed of two links with two sub units.
• The sequence are specialized in the protein for the destined nuclei.

Experiments on Nuclear Protein Import : Antigen T

• Antigen T protein: Large with 90 KD.
• Encoded by the virus genome of SV40.
• Due to its strength the antigen requires an SLS in order to work.
• The sequencing of the antigen requires 8 acid to import into the core.
• It would lead to mutation in which one of these is not in th