🧬 Lecture A: Topics + Nucleus

Spatial and Temporal Organization of Gene Expression

DNA
 Gene locus condensed → inactive
 Gene locus decondensed → active

mRNA
 Diffuse throughout nucleus
 Concentrated at gene loci (site of transcription)
 Found in mobile ribonucleoprotein particles

Protein
 Diffuse throughout cytoplasm (sites of translation)
 Localized to peroxisomes (target organelle)

Protein Synthesis, Trafficking & Organelle Biogenesis

Nucleus
 Site of mRNA transcription

Endoplasmic Reticulum (ER)
 Site of protein synthesis and initial folding

Golgi
 Modifies, sorts, and packages proteins for transport

Transport Vesicle
 Carries proteins between organelles and to the plasma membrane

Lysosome (Vacuole)
 Contains digestive enzymes for macromolecule breakdown

Mitochondrion / Chloroplast
 Sites of energy production (ATP or photosynthesis)

Peroxisome
 Site of oxidative reactions and specialized protein function

Endosomes
 Involved in protein sorting and trafficking

Plasma Membrane / Extracellular Space
 Final destination for secreted or membrane-bound proteins

Internal Organization – Nuclear Pore Complex & Trafficking

Nuclear Pore Complex
 Gateway for molecules between nucleus and cytoplasm

Nucleocytoplasmic Trafficking
 Movement of mRNA, proteins, and other macromolecules through the nuclear pore

Key Point
 The nuclear pore complex controls transport to maintain proper cellular function

Endomembrane System – Overview

Definition
 Dynamic, coordinated network of organelles and related compartments

Trafficking Pathways

Biosynthetic Pathway
 From ER → lysosome

Secretory Pathway
 From ER → plasma membrane / extracellular space

Endocytic Pathway
 From plasma membrane / ECM → lysosome

Key Point
 The endomembrane system organizes protein and membrane trafficking through biosynthetic, secretory, and endocytic pathways

Protein Processing & Quality Control

Morphology & Dynamics
 Structure and organization of organelles involved in protein synthesis and trafficking

Co-Translational Protein Import
 Proteins are imported into organelles while being synthesized

Processing
 Proteins undergo folding, modification, and sorting

Quality Control / Turnover
 Misfolded or damaged proteins are degraded by the proteasome

Key Point
 Cells maintain protein function through co-translational import, processing, and proteasome-mediated turnover

ER to Golgi Transport

ER to Golgi Transport

ER Exit Sites & Vesicle Assembly
 Regions of the ER where proteins and lipids are packaged into transport vesicles

Transport & Fusion
 Vesicles move from ER → Golgi and fuse with Golgi membranes for further processing

Key Point
 ER exit sites and vesicle transport are essential for protein sorting and trafficking between organelles

Golgi Complex – Structure, Function & Trafficking

Morphology
 Stacked cisternae with distinct cis, medial, and trans regions

Function
 Modifies, sorts, and packages proteins for delivery to organelles, plasma membrane, or extracellular space

Protein Trafficking
 Proteins arrive from ER, undergo processing, and are shipped to their final destinations

Key Point
 The Golgi complex is central for protein maturation, sorting, and trafficking

Lysosomes, Endosomes & Clathrin-Coated Vesicles

Lysosomes (Vacuoles)
 Contain digestive enzymes for breaking down macromolecules

Endosomes
 Involved in sorting and trafficking of internalized material

Clathrin-Coated Transport Vesicles
 Vesicles with a clathrin protein coat that mediates transport between organelles and from plasma membrane → endosomes

Key Point
 These compartments and vesicles coordinate protein and membrane trafficking and degradation within the cell

Cellular Transport – Exocytosis & Endocytosis

Exocytosis
 Vesicles fuse with the plasma membrane to release proteins or other molecules outside the cell

Receptor-Mediated Endocytosis
 Specific molecules are internalized via receptors on the plasma membrane

Endosomes
 Sort and direct internalized molecules to their proper cellular destinations

Phagocytosis
 Engulfment of large particles or pathogens into phagosomes for degradation

Key Point
 Cells use exocytosis, endocytosis, and phagocytosis to control intake, processing, and release of materials

Mitochondria & Chloroplasts – Structure, Dynamics & Protein Import

Morphology
 Mitochondria: double membrane, inner membrane folded into cristae
 Chloroplasts: double membrane, internal thylakoid membranes

Dynamics
 Fission, fusion, and movement within the cell to meet energy needs

Protein Import
 Proteins are synthesized in the cytoplasm and imported into organelles via specific translocases

Key Point
 Mitochondria and chloroplasts rely on imported proteins and dynamic morphology for proper energy production and function

Fluorescent Proteins & Live-Cell Imaging

Discovery & Development
 Green Fluorescent Protein (GFP) from jellyfish
 Creation of genetically modified colored variants of GFP

Applications
 Bacteria colonies on agar plates expressing GFP or other fluorescent proteins
 Study of mitochondrial dynamics using photo-switchable GFP (red ↔ green)

Variants
 BFP – Blue Fluorescent Protein
 YFP – Yellow Fluorescent Protein
 Tangerine – Orange variant
 RFP – Red Fluorescent Protein

Key Point
 Fluorescent proteins allow visualization of dynamic processes in live cells using advanced microscopy

Fluorescent Proteins in Living Cells & Organisms

Transformed Organisms
 Bacteria, Yeast, Worm, Fly, Plant, Fish, Cat, Mouse, Human, Primate

Purpose
 Visualize cellular and developmental processes in living cells and organisms

Key Point
 GFP and its variants can be used across a wide range of species to study dynamic biological processes in real time

Nucleus – Structure & Function

Size & Number
 Largest organelle, usually one nucleus per cell
 Size varies between cells and organisms
 Generally correlates with cell (cytoplasmic) volume
 Increases during development and in cancer cells – useful for diagnosis and prognosis

Prokaryotes vs Eukaryotes
 Eukaryotes: have a membrane-bound nucleus
 Prokaryotes: have a nucleoid – less DNA, minimal DNA packaging, limited/no RNA processing

Key Point
 The nucleus stores genetic material and distinguishes eukaryotic from prokaryotic cells

Nucleus – Main Functions

1. Compartmentalization
 Houses the cellular genome and its activities
 Site of DNA replication, transcription, and RNA processing
 Synthesis of translation components: ribosomes, mRNAs, tRNAs

2. Coordination of Cellular Activities
 Controls metabolism, protein synthesis, and cell division

Key Point
 The nucleus organizes genetic material and coordinates essential cellular functions

Nucleus – Gene Expression Regulation

Spatial & Temporal Separation
 Cytoplasm separated from genome enables controlled gene expression in eukaryotes

Prokaryotes vs Eukaryotes
 Prokaryotes: mRNAs are translated during transcription
 Eukaryotes: mRNAs undergo post-transcriptional processing (splicing, etc.) before being transported out of the nucleus for translation in the cytoplasm or at the ER

Additional Control
 Nuclear envelope restricts access of transcription factors from cytoplasm to DNA
 Provides extra regulation of gene expression in eukaryotes

Key Point
 Separation of the nucleus allows more precise temporal and spatial control over gene expression

Nucleus – Nucleoplasm & Nucleolus

Nucleoplasm
 Fluid-filled interior of the nucleus, highly organized
 Contains >30 specialized subdomains for specific functions
 Subdomains are not membrane-bound

Nucleolus
 Most conspicuous nuclear subdomain, dense and granular
 Size and number (1–5) depend on cell metabolic activity – more active cells have larger and more nucleoli
 Function: produces ribosomes
 Site of rDNA transcription, rRNA processing, and initial ribosomal subunit assembly
 Final ribosome assembly occurs in the cytoplasm

Key Point
 The nucleoplasm organizes nuclear functions, while the nucleolus specializes in ribosome production

Nucleus – Chromosome Organization

Chromosomal Subdomains
 Chromosomes are organized into discrete subdomains during interphase
 Gene location often correlates with activity
 Euchromatin (actively transcribed genes) found at periphery of subdomains

Inter-Chromosomal Channels
 Regions between subdomains that prevent unwanted DNA-DNA or DNA-protein interactions

Transcription Factories
 Regions where genes with similar functions cluster to share regulatory elements

Key Point
 Nuclear organization supports efficient and regulated gene expression

Nucleus – Nuclear Speckles

Definition & Function
 Subdomains appearing as ‘speckles’ where mRNA splicing factors are concentrated
 Site of pre-mRNA processing

Location
 Often found in interchromosomal channels near transcription factories

Dynamics
 Numerous and highly dynamic – move, grow/shrink, and change number based on cellular needs
 Increased transcription affects organization and size of speckles in mammalian cells

Key Point
 Nuclear speckles organize splicing machinery and adapt dynamically to support gene expression

Nucleus – Nuclear Envelope

Definition & Function
 Separates nuclear contents (e.g., genome) from cytoplasm
 Acts as a barrier, allowing regulated passage of molecules like RNA and proteins
 Establishes a unique nuclear composition and spatially regulates gene expression
 Provides structural framework for the nucleus

Composition
 Nuclear membranes
 Nuclear lamina
 Nuclear pore complexes

Key Point
 The nuclear envelope maintains nuclear compartmentalization and controls molecule transport