Transcript Bio 402/502 Section II, Lecture 1

Bio 402/502
Section II, Lecture 1
The Cell Nucleus and Its
Organization
Dr. Michael C. Yu
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
Diagram of a Cell’s nucleus
Associated with heterochromatin
?
Splicing factor
localization
(Website of Dr. D. Spector, CSHL)
• Nuclear functions: what are they?
DNA replication, gene expression, etc
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
Nuclear lamina
(Website of Dr. D. Spector, CSHL)
Electron micrograph of nuclear membrane reveals its function
Nuclear membrane:
• Compartmentalize the nucleus
• Constitutes inner membrane (IM)
and outer membrane (OM)
OM: contiguous with rough endoplasmic
reticulum (ER)-protein synthesis.
IM: proteins such as lamins are anchored
to the IM.
(UTMB Cell Biology)
More on nuclear membrane/nuclear envelope
Nuclear lamins are building blocks of
nuclear architecture.
(the Cell website)
Nuclear membrane:
• Studded with nuclear pores
(Voelt et al, 2002)
Functions of lamins
• 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
(Alberts et al)
(slide from Jess Hurt, HMS)
Different types of lamin and organisms that have them
-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 A10
Lamin C
Lamin C2
(Stewart, Curr. Op. Gen. and Dev. 2003)
(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
Differences in the nuclei from lamin-caused disorder
Progeria
Mandibuloacral dysplasia
Normal
(Novelli C., TRENDS in Mol. Med. 2003)
Immunostaining: use of a protein-specific antibody followed by fluorescentdye 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)
Change in the nuclear membrane
architecture
}
WT
(Guillemin et al 2001)
Mutant
Nuclear structure defects due to lamin
Phenotype of LMNA -/- mouse
Continuous
association of
chromatin with
nuclear envelope
Discontinuities in
association of
chromatin with
nuclear envelope
Electron microscopy of wt and Lmna-/- MEF’s
(T. Sullivan et al. J. Cell Biol. 147 (1999) 913-919)
(slide from Jess Hurt, HMS)
Lamin provides anchorage sites for chromatin
Experimental Evidence Supporting Lamin Functions
Provide anchorage sites for chromatin:
• Genetic studies using Drosophila, C. elegans, mouse
(KO of lamin gene)
DAPI (DNA) stain - sees abnormal DNA
organization
}
WT
(Guillemin et al 2001)
Mutant
Nuclear structure defects due to lamin
Laminopathies & Mechanism
How are tissue-specific effects achieved by mutations found in all
cells?
Hypothesis 1: Structural hypothesis
Mutations in lamins predispose all cells to fragility. Muscle cells are
affected most. Falls short in lipodystrophies.
Hypothesis 2: Gene expression hypothesis
Disease phenotype due to alterations in gene expression that affect
particular cell types.
What experiments can one perform to test these hypothesis?
Nuclear Pore Complex
(Website of Dr. D. Spector, CSHL)
The nuclear pores on the membrane
• Purpose of nuclear pores?
-allows for exchange of macromolecules
-NPCs are dynamic
• Type of cargo transported?
-proteins, ribosomes, RNPs,
and RNAs
• How is this achieved?
-Via Nups (proteins of the NPC)
-assembly/disassembly of cargos
via exchange of GDP for GTP by
Ran
EM of nuclear pore complex
The nuclear pore complex: gateway to the nucleus
Bi-directional transport of macromolecules
The Nuclear Pore Complex
Cytoplasmic
filament
Cytoplasm
Cytoplasmic
ring
~2000Å
Nucleus
Inner ring
~150Å
Basket
Ribosome
Distal ring
Nucleo-Cytoplasmic Transport
Ribosomal
Subunits
mRNA
Ribosomal
Proteins
mRNA
The nuclear pore complex: gateway to the nucleus
• Non-static
• All macromolecule transport are energy-dependent
• Gene Gating hypothesis? Functional connectivity with NPC
Will be discussed in detail in lecture #5
Nucleolus
(Website of Dr. D. Spector, CSHL)
Nucleolus: a sub-organelle of the nucleus
• Not membrane-bound
• Function: site of ribosome production, rRNA processing
and synthesis
Nucleolus: a sub-organelle of the nucleus
HeLa Cells’ nucleolus
Isolated
nucleoli
ID nucleolar proteins by mass
spec (approx. 700 proteins)
(Lam et al, 2005)
Chromosome Territories
(Website of Dr. D. Spector, CSHL)
Chromosome Territories
• Individual chromosomes are organized into chromosome
territories (CTs)
• Chromatins are dynamic - interactions with nuclear
architecture
• Correlation between CT structure and function (active vs.
inactive X chromosome)
Purpose: to facilitate/regulate gene expression
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
Cis-interaction/trans
interaction
Cis and trans
co-association
Speckle
Transcription factory
(Fraser & Bickmore, 2007)
Chromatin loop
Correlation between chromosome location and gene expression
Outstanding questions on chromosome territories
How do chromosomes form this higher-order structure?
How do chromosomes find their place in the nucleus?
Mechanism of chromosome positioning?
Thoughts on how chromosome positioning affects gene
expression?
Will be discussed in detail in lecture #6