1. Bio 402/502 Section II, Lecture 1
The Cell Nucleus and Its Organization
Dr. Michael C. Yu

2. 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

3. Nuclear functions: what are they?
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

4. THE MOST IMPORTANT FUNCTION: GENE EXPRESSION
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

5. Nuclear lamina
(Website of Dr. D. Spector, CSHL)

6. Compartmentalize the nucleus
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)

7. Nuclear lamins are building blocks of nuclear architecture.
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)

8. Intermediate filament proteins
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)
Phosphorylation:
+P at end of prophase->disassembly as NE breaks down
-P just as daughter cell forms and lamins reassemble around chromosomes
(slide from Jess Hurt, HMS)

9. 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 C2
(Stewart, Curr. Op. Gen. and Dev. 2003)
(slide from Jess Hurt, HMS)

10. Human diseases due to mutations in the nuclear envelope
(Broers, J. L. V. et al. Physiol. Rev. 86: )

11. Hutchinson-Gilford Progeria syndrome: accelerated aging
Patients suffering from lamin disorder (progeria)
Hutchinson-Gilford Progeria syndrome: accelerated aging

12. 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 fluorescent-dye conjugated secondary antibody to detect the protein-specific antibody bound on a slide/tissue section

13. } Nuclear envelope assembly:
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 }
Nuclear structure defects due to lamin WT
Mutant
(Guillemin et al 2001)

14. (T. Sullivan et al. J. Cell Biol. 147 (1999) 913-919)
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) )
(slide from Jess Hurt, HMS)

15. Lamin provides anchorage sites for chromatin

16. } Provide 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 }
Nuclear structure defects due to lamin WT
Mutant
(Guillemin et al 2001)

17. What experiments can one perform to test these hypothesis?
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?

18. Nuclear Pore Complex
(Website of Dr. D. Spector, CSHL)

19. 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

20. EM of nuclear pore complex

21. Bi-directional transport of macromolecules
The nuclear pore complex: gateway to the nucleus
Bi-directional transport of macromolecules

22. The Nuclear Pore Complex
Cytoplasmic
filament
Ribosome
~150Å
~2000Å
Cytoplasmic
ring
Cytoplasm
Nucleus
Inner ring
Basket
Distal ring

23. Nucleo-Cytoplasmic Transport
Ribosomal
Subunits
Proteins
mRNA

24. All macromolecule transport are energy-dependent
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

25. Nucleolus
(Website of Dr. D. Spector, CSHL)

26. Function: site of ribosome production, rRNA processing and synthesis
Nucleolus: a sub-organelle of the nucleus
Not membrane-bound
Scan Cell Fig 6-47
Function: site of ribosome production, rRNA processing and synthesis

27. Nucleolus: a sub-organelle of the nucleus
HeLa Cells’ nucleolus
Isolated nucleoli
Scan Cell Fig 6-47
ID nucleolar proteins by mass spec (approx. 700 proteins)
(Lam et al, 2005)

28. Chromosome Territories
(Website of Dr. D. Spector, CSHL)

29. 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

30. Distribution of transcription sites in relations to CTs
Correlation between chromosome territories & gene activity
(Verschure et al, 1999)
Distribution of transcription sites in relations to CTs

31. Correlation between chromosome location and gene expression
Colocalization of genes in the nucleus for expression or coregulation
Cis-interaction/trans
interaction
Cis and trans
co-association
Speckle
(Fraser & Bickmore, 2007)
Transcription factory
Chromatin loop
Correlation between chromosome location and gene expression

32. How do chromosomes form this higher-order structure?
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