Friday, April 23, 2010

More about the Nucleus: Matrix, Envelope, Pores, Lamins

I needed that little break.  Exams have calmed temporarily and I have begun studying in earnest yet again.  Now I plan to take a slightly different approach with these updates by moving through the material chronologically, as it was taught.  Maybe using this method, I'll be able to refer back  and interlink posts more efficiently.
The Nuclear Matrix
As mentioned, DNA wrapped in nucleosomes loops and attaches to the nuclear matrix, but what exactly is the nuclear matrix?  Technically, the nuclear matrix consists of what is left after the DNA, lipid, and protein content of the nucleus has been cleared.  It consists of the nucleolus, the nuclear pore complex and lamina, and the internal nuclear matrix.  Many have argued that the nuclear matrix is simply an artifact of the extraction process.

The nucleolus in the nuclear matrix consists of ten chromosomes that converge to form a small compartment.  These ten chromosomes all contain genes for rRNA, and the nucleolus is where the rRNA is synthesized.  The ribosomal rRNA is synthesized by RNA pol I and III, and after synthesis, proteins are added to the rRNA while it is still in the nucleus.  Various subcompartments of the nucleolus have also been identified: the fibrillar center consists of the nucleolar organizer and the rDNA genes; the dense fibrillar (pars fibrosa) consists of the sites of transcription; and the granular (pars granulose) makes up the ribosome subunits. 

The Nuclear Envelope and Pore Complex Lamina
The nuclear envelope consists of a double membrane that connects to the ER on the outside and to the nuclear lamina (and heterochromatin) on the inside.  The lamin B receptor (LBR) can be found in the nuclear envelope, as it is an integral membrane protein.  Also contained in the envelope is the nuclear pore complex.  The NPC is from 50-to-150 nm in size, with about 5,000 found on the membrane per nucleus.  At the NPC is where the inner and outer membranes of the nuclear envelope come together, and the NPC is involved in communication between chromatin and events outside the nucleus. 

The nuclear pore complex can pass 500 macromolecules per second, with molecules less than 5000 Da passing freely; those macromolecules larger than 60 kDa barely enter.  The channel that allows passage of these molecules is only approximately 10 nm wide.  The complex itself is made of 1000 proteins called nucleoporins, forming an octomeric structure.  It functions to import nuclear proteins via nuclear localization sequences (NLSs), a protein sequence that signals nuclear import.  A protein with more NLSs is imported more frequently, though an NLS does not mediate retention of the protein.  The protein importin, made of α and β subunits, assists in protein import by binding the NLS.  The importin receptor recognizes the NLS and then migrates on FG repeats of the nucleoporins to dock and translocate through the pore.  The cargo (with its NLS) is then released to the nucleus. 
Export of mRNA and proteins from the nucleus is also important to the cell.  It has been discovered that the sequence of the RNA does not affect its ability to export from the nucleus where it originated.  However, it has also been discovered that different RNAs are exported via different pathways, which may be facilitated by RNA binding proteins that contain a nuclear export sequence (NES).  Exports are the receptors for these NESs and facilitate protein movement out of the nucleus.  Those proteins with both an NES and NLS are considered shuttle proteins. 

Nuclear import and export is heavily regulated in the cell.  Phosphorylation can affect nuclear import: direct phosphorylation o the NLS can inhibit transport.  Ran-GTP is another important factor that affects transport.  Ran-GTP in the nucleus binds to empty receptors and transport them to the cytoplasm, where they can reload with a piece of cargo.  Ran-GAPs in the cytoplasm facilitate the hydrolysis of GTP to GDP, which promotes translocation of Ran-GDP to the nucleus.  Once in the nucleus, Ran-GEFs promote the exchange of GDP for GTP.  The interaction of the different forms of Ran allow for the recycling of shuttling proteins, allowing them to move proteins in and out of the nucleus.

The Nuclear Lamina
As mentioned, the nuclear envelope surrounds the nucleus and connects to the lamina on its inner face.  The nuclear lamina itself is about 75 nm thick and is composed of proteins similar to intermediate filaments, called lamins.  Lamins come in three forms: A, B, and C.  Lamins A and C bind heterochromatin.  Lamin B binds the lamin B receptor (LBR), which is connected to the nuclear envelope as an integral membrane protein.  The lamin complexes also peripherally bind to the NPC.  Lamins maintain the nucleus in its spherical shape, and phosphorylation of A and C subunits solubilizes them during prophase, allowing dissolution of the nuclear lamina.  Lamin B remains attached to its receptor during prophase, however.  Importantly, mutations in these proteins can cause laminopathies because they are involved in nuclear organization, and mutations can result in inhibited DNA synthesis.

The Inner Nuclear Matrix
Study of the inner nuclear matrix has shown that the protein composition is cell-type specific.  Additionally, chromosomes are not positioned randomly in the nucleus.  This has been exported by FISH using whole-chromosome probes.  It appears that chromosomes occupy specific territories and, at least in yeast, they take the Rabl conformation, with the telomeres and centromeres directly interacting with the nuclear lamina.  Matrix proteins function in both replication and transcription, and mRNAs from active genes can be found in the matrix, as can newly replicated DNA.  Additionally, matrix proteins of cancerous cells are different from normal cells.

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