bio 402/502 section ii, lecture 1 the cell nucleus and its organization dr. michael c. yu
TRANSCRIPT
Outline of lectures by Dr. Yu (Section II)
Lecture 1: The Cell Nucleus: an overview
Lecture 2: Nuclear Processes: DNA replication/transcription
Lecture 3: Nuclear Processes: Transcription/mRNA splicing
Lecture 4: Nuclear Processes: mRNA processing and export
Lecture 5: Nuclear protein transport
Lecture 6: Chromosome Territory & Nuclear organization
Lecture 7: Systems biology of the nucleus
Exam Q’s: materials from assigned primary articles, lectures, and some textbook readings (available for copying in the Biological
Sciences Dept Office) Exam Format: open book & open notes/journal articles
• Nuclear functions: what are they?
Diagram of a Cell’s nucleus
(Website of Dr. D. Spector, CSHL)
DNA replication, gene expression, etc
Associated with heterochromatin
Splicing factor localization
?
Nuclear Functions Revealed
• Nuclear envelope: provides the compartmentalization and structure
• Nucleolus: site of snRNA and ribosomal RNA maturation
• Heterochromatin: role in gene expression
•Chromosomal territory (CT): higher order organization
THE MOST IMPORTANT FUNCTION: GENE EXPRESSION
Electron micrograph of nuclear membrane reveals its function
Nuclear membrane:• Compartmentalize the nucleus• Constitutes inner membrane (IM) and outer membrane (OM)
(UTMB Cell Biology)
OM: contiguous with rough endoplasmic reticulum (ER)-protein synthesis.
IM: proteins such as lamins are anchored to the IM.
More on nuclear membrane/nuclear envelope
Nuclear membrane:• Studded with nuclear pores
(the Cell website)
(Voelt et al, 2002)
Nuclear lamins are building blocks of nuclear architecture.
(Alberts et al)
• Intermediate filament proteins• Form meshwork at inside of inner nuclear membrane (INM), some extend into nucleoplasm• Nuclear strength and architecture• DNA replication and mRNA transcription• Involved in apoptosis
Functions of lamins
(slide from Jess Hurt, HMS)
-Lamins are only found in nuclei of multicellular eukaryotes
Organism Lamins
Yeast -
Worms LMN-1
Fly Lamin C
DmO
Humans Lamin B1
Lamin B2
Lamin B3
Lamin A
Lamin A10
Lamin C
Lamin C2(Stewart, Curr. Op. Gen. and Dev. 2003)
Different types of lamin and organisms that have them
(slide from Jess Hurt, HMS)
Human diseases due to mutations in the nuclear envelope
(Broers, J. L. V. et al. Physiol. Rev. 86: 967-1008 2006)
Patients suffering from lamin disorder (progeria)
Hutchinson-Gilford Progeria syndrome: accelerated aging
Progeria Mandibuloacral dysplasia Normal
(Novelli C., TRENDS in Mol. Med. 2003)
Differences in the nuclei from lamin-caused disorder
Immunostaining: use of a protein-specific antibody followed by fluorescent-dye conjugated secondary antibody to detect the protein-specific antibody bound on a slide/tissue section
Experimental Evidence Supporting Lamin Functions
Nuclear envelope assembly:
• Genetic studies using Drosophila, C. elegans, mouse (KO of lamin gene)
Nuclear structure defects due to lamin
WT Mutant
}(Guillemin et al 2001)
Change in the nuclear membrane architecture
Electron microscopy of wt and Lmna-/- MEF’s(T. Sullivan et al. J. Cell Biol. 147 (1999) 913-919)
Discontinuities in association of chromatin with nuclear envelope
Continuous association of chromatin with nuclear envelope
(slide from Jess Hurt, HMS)
Phenotype of LMNA -/- mouse
Experimental Evidence Supporting Lamin Functions
• Genetic studies using Drosophila, C. elegans, mouse (KO of lamin gene)
Nuclear structure defects due to lamin
WT Mutant
}(Guillemin et al 2001)
DAPI (DNA) stain - sees abnormal DNA organization
Provide anchorage sites for chromatin:
How are tissue-specific effects achieved by mutations found in all cells?
Hypothesis 1: Structural hypothesisMutations in lamins predispose all cells to fragility. Muscle cells are affected most. Falls short in lipodystrophies.
Hypothesis 2: Gene expression hypothesisDisease phenotype due to alterations in gene expression that affect particular cell types.
Laminopathies & Mechanism
What experiments can one perform to test these hypothesis?
The nuclear pores on the membrane
• Type of cargo transported?
• How is this achieved?
• Purpose of nuclear pores?
-allows for exchange of macromolecules
-NPCs are dynamic
-proteins, ribosomes, RNPs, and RNAs
-Via Nups (proteins of the NPC)
-assembly/disassembly of cargos via exchange of GDP for GTP by Ran
CytoplasmicCytoplasmicfilamentfilament
CytoplasmCytoplasm
NucleusNucleus
CytoplasmicCytoplasmicringring
Inner ringInner ring
BasketBasket
Distal ringDistal ring
The Nuclear Pore ComplexThe Nuclear Pore Complex
RibosomeRibosome
~150Å
~2000Å
mRNAmRNA
mRNAmRNA
RibosomalRibosomalSubunitsSubunits
RibosomalRibosomalProteinsProteins
Nucleo-Cytoplasmic TransportNucleo-Cytoplasmic Transport
The nuclear pore complex: gateway to the nucleus
• All macromolecule transport are energy-dependent
• Non-static
Will be discussed in detail in lecture #5
• Gene Gating hypothesis? Functional connectivity with NPC
Nucleolus: a sub-organelle of the nucleus
• Function: site of ribosome production, rRNA processing and synthesis
• Not membrane-bound
Nucleolus: a sub-organelle of the nucleusHeLa Cells’ nucleolus
Isolated nucleoli
ID nucleolar proteins by mass spec (approx. 700 proteins)
(Lam et al, 2005)
Chromosome Territories
• Individual chromosomes are organized into chromosome territories (CTs)
Purpose: to facilitate/regulate gene expression
• Chromatins are dynamic - interactions with nuclear architecture
• Correlation between CT structure and function (active vs. inactive X chromosome)
Correlation between chromosome territories & gene activity
(Verschure et al, 1999)
Distribution of transcription sites in relations to CTs
Colocalization of genes in the nucleus for expression or coregulation
(Fraser & Bickmore, 2007)
Correlation between chromosome location and gene expression
Cis and transco-association
Cis-interaction/transinteraction
Speckle
Chromatin loopTranscription factory