Bio 402/502Section 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

(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

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)

(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

Lamin provides anchorage sites for chromatin

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?

Nuclear Pore Complex

(Website of Dr. D. Spector, CSHL)

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

EM of nuclear pore complex

The nuclear pore complex: gateway to the nucleus

Bi-directional transport of macromolecules

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

(Website of Dr. D. Spector, CSHL)

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

(Website of Dr. D. Spector, CSHL)

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

Outstanding questions on chromosome territories

How do chromosomes find their place in the nucleus?

How do chromosomes form this higher-order structure?

Will be discussed in detail in lecture #6

Mechanism of chromosome positioning?

Thoughts on how chromosome positioning affects gene expression?