Nuclear Structure

Why is it hard to isolate the nuclear envelope using cell fractionation?

Because the nuclear envelope is continuous with the ER, so they sometimes sediment together and the pellet is not homogenous

How has our ability to study the nucleus increased in the past few years?

Using super-high resolution microscopy techniques

Do we have a complete understanding of the physical properties (ex. ion concentration, viscosity, etc.) of the nucleoplasm?

No

What is the nuclear lamina?

It is a mesh right below the inner nuclear membrane that is formed by lamin filaments

What is the nucleoplasmic reticulum and what is its function?

It is extensions of the nuclear envelope; the nuclear envelope can invaginate inwards into the nucleoplasm; this is often an extension of the inner nuclear membrane, but both the inner and outer membranes can also invaginate and pinch off to form an “island” of cytoplasm; the nucleoplasmic reticulum can contain specific proteins on its membrane; functions of the nucleoplasmic reticulum include: form a platform for signaling molecules to signal events in the nucleus (ex. kinases and phosphotases), site of lipid synthesis for lipids needed in the nucleus, store Ca²⁺ ions

Do all cells display a nucleoplasmic reticulum?

No, it depends on the cell type and state

What are the functions of the nuclear envelope?

• Compartmentalization → barrier between nucleoplasm and cytoplasm; contains unique populations of molecules

• Transport → nuclear pore complexes

• Platform for signaling

• Structural Integrity → nuclear lamina

• Regulates Gene Expression

What is the primary structural support for the nuclear envelope?

The nuclear lamina

What happens to the shape of the nucleus if mutant lamin protein is added to a cell?

The shape of the nucleus changes because it loses its support

What is the cause of progeria?

It is a disease of rapid aging where the cell nuclei are very irregularly shaped and this affects gene expression

What is chromatin?

DNA associated with proteins

In what form is DNA typically found in the nucleus?

It is almost always associated with proteins → this is chromatin

What are histones?

They are octomeric complexes (8 subunits) around which DNA will wrap

What is a nucleosome?

DNA wrapped around a histone; about 10 nm in diameter

What are the levels of organization of DNA in the nucleus?

Naked DNA → Nucleosome → Chromatin fibers → Chromatin territory/compartment

What are chromatin fibers?

Nucleosomes associated with each other; they are 30 nm in diameter

What is the transcriptional state of DNA when it is packaged in chromatin fibers?

It is transcriptionally inactive because it is very tightly packaged so the transcription proteins cannot access it

What is the top level of structure of DNA?

A chromatin compartment/territory

When do we see condensed chromosomes?

During mitosis/meiosis

What is a chromatin compartment/territory?

The highest order structure of DNA in a cell; the chromatin fibers of one chromosome associate together in a sort of spaghetti like ball that occupies a certain part of the nucleus; this is the state that DNA is in when cells are not undergoing mitosis/meiosis

What led to the first ideas of there being “stringy material” (chromatin) in the nucleus?

• Carl Rabl looked at salamander epithelial cells under a microscope and saw string-like material with threads coming off of it

What led to the first idea of the nucleus not being homogenous/the existence of chromosome territories?

• Boveri looked at round worm cells and noticed that the nucleus had dense protrusions that stained differently when he used different stains

Is the nucleus homogenous?

No

What did early EM show in the nucleus?

It made it look like the nucleus was homogenous

What experiment showed that decondensed chromosomes occupied specific territories in the nucleus rather than being intertwined and spread out with other chromosomes?

Researchers damaged the DNA with microbeam radiation; if the chromosomes are randomly spread out, then the damage would be observed on multiple chromosomes; if the chromosomes are occupying specific territories, then the damage would only be observed on one or two chromosomes; the cell will repair the radiation damage, so the researchers supplied the cells with radioactive thymine, so regions that underwent damage will incorporate this radioactive nucleotide into the repaired DNA; they used EM autoradiography to see the radioactivity and observed that it was only found in a specific region in the nucleus, giving evidence for chromosome territories

What technique do we use to look at where chromosomes are in the modern day?

Fluorescence in situ hybridization (FISH)

How does FISH work?

It uses a fluorescently-labeled small nucleic acid sequence that is complementary to DNA in the chromosome of interest, so it will match up with the chromosome and highlight where it is in the nucleus; sometimes called chromosome painting

What state are chromosomes in when the cell is not dividing?

In a decondensed state in chromatin territories

What is heterochromatin vs. euchromatin?

• Functional definition:

  • heterochromatin → transcriptionally inactive

  • euchromatin → transcriptionally active

• Cytological definition:

  • heterochromatin → denser regions that stain darkly in EM

  • euchromatin → less dense regions that are lighter in EM

• Structural definition

  • heterochromatin → more compact

  • euchromatin → less compact

Is packing of chromosomes the same as compaction of chromosomes?

No, packing refers to the order/level of organization the DNA is in (nucleosome, chromatin fiber, etc.) whereas compaction just refers to how close together the DNA is within a certain level of organization

Can there be both heterochromatin and euchromatin in one chromosome territory?

Yes

Where does transcription occur?

It occurs in the euchromatin, but not all over; it occurs in specific regions of the nucleus called transcriptional hubs; this means that gene expression is only found in certain spatial locations in the nucleus and the position of a gene changes when it is transcribed (position effects)

Where would you expect to find a gene that is on in the nucleus?

In euchromatic regions in a transcriptional hub

How does DNA move around the nucleus?

Through a form of diffusion; not necessarily random diffusion

What drives nuclear organization?

• The “polymerization” of DNA due to interactions between the protein molecules associated with the DNA (histones and other proteins)

• Homotypic clustering; like molecules associate with like molecules; heterochromatin associates with heterochromatin and euchromatin associates with euchromatin

What constrains nuclear organization?

• Nuclear lamina → proteins on the lamina interact with proteins on the chromatin, physically attaching chromatin to the lamina, which restrains gene expression

• Nuclear bodies → biomolecular condensates that hold on to strands of DNA and fix their position, restraining gene expression

What is the probable transcriptional state of genes close to the nuclear envelope?

These genes are generally not transcribed, as they are often attached to the nuclear lamina

What is the probable transcriptional state of genes further from the nuclear envelope?

These genes are more likely to be transcriptionally active because the nuclear envelope is not associating with them

What is the probable transcriptional state of genes close to a nuclear pore complex?

These genes are generally being transcribed even though they are close to the edge of the nucleus

What are some examples of nuclear bodies?

• Nucleolus → rRNA production/ribosome subunit assembly

• Cajal body → assembly of complexes required for mRNA processing

• Speckles → storage of complexes required for mRNA processing

Does gene expression drive the organization of the nucleus or does the structure of the nucleus drive gene expression?

Gene expression drives the organization of the nucleus; once organized, the structure of the nucleus regulates and constrains gene expression