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

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

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

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

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)

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)

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)

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)

Human diseases due to mutations in the nuclear envelope (Broers, J. L. V. et al. Physiol. Rev. 86: 967-1008 2006)

Hutchinson-Gilford Progeria syndrome: accelerated aging 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 fluorescent-dye conjugated secondary antibody to detect the protein-specific antibody bound on a slide/tissue section

} 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)

(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) 913-919) (slide from Jess Hurt, HMS)

Lamin provides anchorage sites for chromatin

} 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)

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?

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

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

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

Nucleo-Cytoplasmic Transport Ribosomal Subunits Proteins mRNA

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

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

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

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)

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

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

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

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

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