Cell Export - Exocytosis

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Exocytosis and Endocytosis cartoon

Contents

Introduction

This lecture introduces how information is transferred from stable stored information (DNA) converted to an intermediate (mRNA, rRNA, tRNA) of variable stability, exported from the nucleus to the cytoplasm where mRNA is then translated into Protein. This is gene expression, the products of this process are used either within the cell, exported (exocytosis) or used to replace worn out components.

We will study this topic at the level of the cellular components and organelles involved in the process: ribosomes, endoplasmic reticulum, Golgi apparatus, vesicles (transport and secretory).


Note external lecture links to Bookshelf Cell Biology textbooks and JCB have changed. Links should automatically forward, some may not function. I am currently updating these links as we go through 2013 lecture series.
Exocytosis movie 1.jpg
 ‎‎Exocytosis
Page | Play

Lecture Audio

The University has a system for automated recording of lectures called Lectopia. Lectopia requires login using your student number and unipass. I will be adding the link to each iLecture Audio following the Lecture. Due to the automated recording method, most lectures begin 4-5 minutes into MP3 recordings and occasionally stop before the lecture does.


Lecture 5 Audio Lecture Date: 2013-03-26 Lecture Time: 15:00 Venue: WW LG02 Speaker: Dr Mark Hill


Archive: 2012 | 2012 Print version | 2010 | 2009

MH - note that content listed below will not match exactly current lecture structure but has been selected as having similar content.

Objectives

  • Brief understanding of gene expression
  • Understanding structure and function of organelles and structures associated with protein export (exocytosis)
    • ribosome
    • endoplasmic reticulum
    • vesicles - types and transport
    • Golgi apparatus structure and function
  • Brief understanding of transport between secretory compartments
  • Brief understanding of membrane turnover

History

Ribosomes first EM

Below are some example historical research finding related to exocytosis from the JCB Archive.

1955 Ribosomes, or the particles of Palade George Palade identifies particulate components of the cytoplasm, known initially as the particles of Palade and later as ribosomes.

1956 Microsomes are the in vitro ER George Palade and Philip Siekevitz unite the fields of microscopy and fractionation in this work. They conclude that Albert Claude’s biochemical fraction called microsomes are the in vitro version of the endoplasmic reticulum (ER) — a cytological feature first noted by Keith Porter.

1958 A pathway for secretion Radioactive proteins are followed after their synthesis as they progress towards their secretory fate; this allows the definition of not only trafficking pathways but of the organelles that lie along that pathway.

1966 Excess secretory products fuse with lysosomes Robert Smith and Marilyn Farquhar find that excess secretory granules are not stored but fuse with multivesicular bodies (MVBs) that then mature and fuse with lysosomes.

1975 Lost in translation: the signal hypothesis Günter Blobel and Bernhard Dobberstein use a Rube Goldberg concoction of mouse RNA, rabbit ribosomes, and dog ER to reconstruct cell biology's version of the ship in the bottle: how proteins a cell intends to secrete end up in the endoplasmic reticulum.

2009 The Nobel Prize in Chemistry 2009 awarded to Drs Venkatraman Ramakrishnan, Thomas A. Steitz and Ada E. Yonath "for studies of the structure and function of the ribosome".

Looking in the Cytoplasm

Difference between Prokaryotes and Eukaryotes

  • Light microscope - histology, immunohistochemistry
    • lacks details within cytoplasmic compartment
    • Immunochemistry
    • Organelle dyes
    • Fluorescent tagged proteins
  • Electron microscope
    • shows the organelles and membrane structure

Links: MCB - The secretory pathway of protein synthesis and sorting.

The Cytosol

Cell Protein Distribution
  • Membrane bound compartment
  • About 1/2 total cell volume
  • Intermediary metabolism takes place in the cytosol
    • Chemical biological reactions
    • Degradation
    • Synthesis
  • Protein molecules
    • cell has about 10 billion (1x1010)
    • 10,000 to 20,000 different kinds

Compartments are Dynamic

  • Membrane bound compartments change shape and size
  • Related to cell cycle, differentiation, signaling

Proteintransport.gif


Links: Overview of sorting of nuclear-encoded proteins in eukaryotic cells | MBoC Table 12-3. Some Typical Signal Sequences

Ribonucleic Acid (RNA)

  • 3 types of RNA
    • Messenger RNA (mRNA) translated into protein by action of ribosomes
    • Transfer RNA (tRNA) each tRNA is specific for a specific amino acid (anti-codon)
    • Ribosomal RNA (rRNA) Forms the backbone of ribosome subunits

Messenger RNA (mRNA)

  • processed in the nucleus
  • biological molecule (polymer) with unstable (half-life)
  • exported to the cytoplasm
  • site of ribosome assembly

Messenger RNA movie

Transfer RNA (tRNA)

  • small RNA molecules which each binds a specific amino acid (anti-codon) tRNA
  • carries it to the ribosome for protein assembly

Ribosomal RNA (rRNA)

Ribosomes - Prokaryotic and Eukaryotic
  • provide framework for dozens of proteins involved in assembly of AA sequence into protein
  • most abundant RNA in cells
  • Usually characterized by sedimentation coefficient
    • 40S & 60S
  • rRNA genes are located in nucleolus
  • multiple copies of human ribosomal RNA genes (rDNA) are arranged as tandem repeat clusters on the middle of the short arms of chromosomes 13, 14, 15, 21, and 22. PMID: 3336775

Links: MCB - Overview of mRNA processing in eukaryotes | MCB - movie - Life Cycle of an mRNA

Ribosomes

Translation movie 1.jpg
 ‎‎Translation
Page | Play

Ribosome Structure

Ribosomes - Prokaryotic and Eukaryotic
  • Ribosome biogenesis consumes up to 80% of the energy of the cell PMID 10806485
  • All RNA components mRNA, rRNA and tRNA come together in this structure
  • two ribosome types with identical structure
  • different locations - free and membrane bound
    • Free in cytoplasm
    • Bound to endoplasmic reticulum
  • Also located within mitochondria

Ribosome Function

  • Protein Synthesis
  • complexes where RNA sequences are converted to amino acid (aa) sequences
  • Codons 3 NTPs = 1 AA
    • AA incorporated at 20/sec
    • average sized protein takes 20-60 seconds to assemble
  • Synthesis from amino- to carboxy- terminal of protein
  • many ribosomes can bind 1 mRNA (polyribosome)
  • signal recognition particle (SRP) - ribonucleoprotein (protein-RNA complex) that recognizes and targets proteins to the endoplasmic reticulum

Polyribosomes

  • polyribosomes or polysomes are the EM visible granules
  • many ribosomes bound to a single mRNA
  • single ribosome covers a 54bp mRNA region
  • the synthesised single amino acid chain can then be "modified"
    • in the cytoplasm or in specialised organelles
  • Protein Modification/Function


Links: MBoC - Figure 1-10. A ribosome at work | MBoC - Figure 12-37. Free and membrane-bound ribosomes | MBoC - Figure 6-63. A comparison of the structures of procaryotic and eucaryotic ribosomes | MCB - Model of protein synthesis on circular polysomes and recycling of ribosomal subunits (movie)

Endoplasmic Reticulum

Nucleus and RER tem
  • endoplasmic ‚"within the cell"
  • reticulum ‚ "a little net"
  • an organelle, membrane bound compartment. within the cytoplasmic space
  • One structural compartment
  • Two functional compartments
    • Rough Endoplasmic Reticulum (RER)
    • Smooth Endoplasmic Reticulum (SER)
Endoplasmic reticulum movie 1.jpg
 ‎‎Endoplasmic Reticulum
Page | Play
Mammalian proteins transported into endoplasmic reticulum
Er tubular domains

Links: MBOC - The Endoplasmic Reticulum

Rough Endoplasmic Reticulum

Rough Endoplasmic Reticulum - Function

Protein Cellular Transport/Targeting
Mammalian proteins transported into er
  • Allows specific proteins to be modified and targeted to different destinations
  • Modification
    • amino acid chain cleaved or sidegroups added (mainly glycosylation)
    • glycosylation = addition of carbohydrate (sugar) groups
  • Destination
    • Domestic - Cytosolic, Nuclear, Organelles
    • Exported from cell


Links: MCB - Overview of sorting of nuclear-encoded proteins in eukaryotic cells | MCB - movie - Protein Sorting JCB- movie - Real-time video of the formation of tubules at ER export sites

Rough Endoplasmic Reticulum - Structure

  • about 50% of cell membrane
  • continuous with outer nuclear membrane
  • single highly convoluted membrane enclosing a single space
  • ER lumen = ER cisternae
  • "rough" because of many ribosomes attached to the membrane
  • ribosomes bound only to cytoplasmic side of ER membrane
Nucleus and RER tem
Interphase ER tomography


Links: MBOC - The Endoplasmic Reticulum JCB - movie - Three-dimensional view of Sar1 tubules

Smooth Endoplasmic Reticulum (SER)

Smooth Endoplasmic Reticulum - Structure

  • Part of same membrane as RER
    • may also be called "transitional"
  • no attached ribosomes
  • not involved in protein synthesis
  • differ in shape
  • SER a meshwork of fine tubules

Smooth Endoplasmic Reticulum - Function

  • lipid metabolism (membrane)
  • carbohydrate metabolism
  • detoxification of drugs and harmful compounds
  • steroid synthesis and metabolism (cholesterol)
  • different amounts in different cells

In muscle cells SER stores and releases calcium ions (Ca2+} to trigger muscle contractions.

(Movie: RER to Golgi)

Links: MBoC - Transport from the ER through the Golgi Apparatus

Transport Vesicles

  • RER synthesized material is transferred by budding off of membrane
  • Forms transport vesicle
  • Transports substances to different cellular locations
  • Most transport to Golgi apparatus
  • Active transport mainly along microtubules (cytoskeleton)

Links: MBoC - Vesicular Traffic | JCB - movie - transport vesicles and lipid (large 9.7 Mb)

Golgi Apparatus

Camillo Golgi

Golgi Apparatus - History

  • Discovered over 100 years ago
  • Camillo Golgi (1898)
    • seen in neurons as anastomosing threads
    • ‚Äúinternal reticular apparatus"
  • Soon detected in many cells
  • Nobel Prize1906 Camillo Golgi, Santiago Ramon y Cajal


Links: MBOC - Golgi Apparatus- Summary

Golgi Apparatus - Structure

Tem golgi1.jpg Tem golgi3.jpg

Golgi structure cartoon
  • organelle, membrane enclosed structural compartment
  • cell may contain one or more Golgi apparatus
  • located near the nucleus
  • disc shaped membrane stack with different regions by their location within the cell

Golgi stack

  • from 6-30/stack
  • 3-100s stacks/cell
  • many sets of membrane bound smooth surfaced cisternae

Stack Nomenclature

  • cis - bottom of stack closest to endoplasmic reticulum, receives transport vesicles from ER
  • medial - middle of stack, processing of proteins, modification of sidechains
  • trans - top of stack closest to plasma membrane, buds off secretory vesicles

  • Golgi Apparatus Electron Micrograph

  • A Golgi from the unicellular green alga Ostreococcus tauri imaged by electron cryotomography


Links: MBoC - Golgi Apparatus | MBoC - Figure 13-30. Two possible models explaining the organization of the Golgi apparatus and the transport of proteins from one cisterna to the next | MCB - Figure 5-49. Three-dimensional model of the Golgi

Golgi Apparatus - Functions

Golgi and centrosome
Golgi fragments during mitosis
  • Sorting of cytosolic/secreted proteins
  • Glycosylation of secreted proteins
  • Modification of carbohydrates
  • Side chains are also trimmed
  • Trans vesicles fuse with the plasma membrane

Post-Golgi transport cartoon.jpg


Golgi and centrosome during cell cycle.jpg

Golgi and centrosome during cell cycle

Secretory Vesicles

Exocytosis types
Docked secretory vesicles
  • protein export (secretion)
    • constitutive and regulated
  • related also to membrane turnover
    • new lipid
    • new cholesterol
    • new membrane proteins


Exo endo cytosis.jpg

Vesicle Movies

Exocytosis movie 1.jpg
 ‎‎Exocytosis
Page | Play
Post-Golgi carriers-icon.jpg
 ‎‎Exocytosis
Page | Play


Links: JCB - movie - Insulin Secretion (3.4 Mb) | JCB - movie - transport vesicles and lipid (9.7 Mb) |

Abnormalities


Links: NCBI - Genes and Diseases | NCBI - OMIM |

References

Textbooks

Essential Cell Biology

  • Chapter 14 Intracellular Compartments and Transport

Molecular Biology of the Cell

Alberts, Bruce; Johnson, Alexander; Lewis, Julian; Raff, Martin; Roberts, Keith; Walter, Peter New York and London: Garland Science; c2002

Molecular Cell Biology

Lodish, Harvey; Berk, Arnold; Zipursky, S. Lawrence; Matsudaira, Paul; Baltimore, David; Darnell, James E. New York: W. H. Freeman & Co.; c1999


The Cell- A Molecular Approach

Cooper, Geoffrey M. Sunderland (MA): Sinauer Associates, Inc.; c2000

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Books

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  • Online Mendelian Inheritance in Man (OMIM) is a comprehensive compendium of human genes and genetic phenotypes. The full-text, referenced overviews in OMIM contain information on all known mendelian disorders and over 12,000 genes. OMIM
  • Entrez is the integrated, text-based search and retrieval system used at NCBI for the major databases, including PubMed, Nucleotide and Protein Sequences, Protein Structures, Complete Genomes, Taxonomy, and others Entrez

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Reviews

  • Lipid rafts and the regulation of exocytosis. Salaün C, James DJ, Chamberlain LH. Traffic. 2004 Apr;5(4):255-64. Review. PMID: 15030567

Articles

Vesicle association and exocytosis at ribbon and extraribbon sites in retinal bipolar cell presynaptic terminals. Zenisek D. Proc Natl Acad Sci U S A. 2008 Mar 25;105(12):4922-7. Epub 2008 Mar 13. PMID: 18339810

A high-throughput screening of genes that encode proteins transported into the endoplasmic reticulum in mammalian cells. Ozawa T, Nishitani K, Sako Y, Umezawa Y. Nucleic Acids Res. 2005 Feb 24;33(4):e34.PMID: 15731327


Cell Structure Images

The linked pages below currently contain unlabeled electron micrographs showing specific cellular features.


JCB Movies

  • A myosin V moves yeast secretory vesicles Secretory vesicles actively move to the site of exocytosis in yeast. Schott et al. find that multiple secretory vesicles often follow the same linear track and frequently enter and cross the bud. This movement requires the activity of the myosin-V heavy chain encoded by the MYO2 gene. When the predicted lever arm of this motor is progressively shortened (with the most extreme example being the 0IQ mutant), the vesicle movements are progressively slowed.
  • Rapid cycling of lipid rafts to and from the Golgi Nichols et al. detect rapid cycling of lipid raft markers between the plasma membrane and the Golgi. Through selective photobleaching, they are able to study transport either out from the Golgi to the plasma membrane, or in from the plasma membrane to the Golgi.
  • Membrane docking at the immunological synapse requires Rab27a Stinchcombe et al. find that normal membrane docking of lytic granules at the immunological synapse is defective in cells lacking Rab27a. In cells lacking other Rab proteins, polarization of the secretory granules is incomplete.
  • Visualizing the location and dynamics of exocytosis Schmoranzer et al. use total internal reflection (TIR) fluorescence microscopy to visualize exocytosis in mammalian cells (e.g., see event on left side of video). The analysis reveals that there are no preferred sites for constitutive exocytosis in this system.
  • Visualizing the location and dynamics of exocytosis Toomre et al. use a combination of TIR microscopy (green, labeling molecules close to or at the membrane) and standard fluorescence microscopy (red, for molecules further from the membrane) to visualize [/content/vol149/issue1/images/data/33/DC1/Fig_1b.mov trafficking to and fusion with] the plasma membrane during exocytosis. Red dots turn yellow then green as they approach the membrane, and then explode in a burst of light as they fuse with the plasma membrane during exocytosis. The transport containers appear to be partially anchored at the membrane before fusion, and can undergo either partial or complete fusion events.


2012 Course Content

Lectures: Cell Biology Introduction | Cells Eukaryotes and Prokaryotes | Cell Membranes and Compartments | Cell Nucleus | Cell Export - Exocytosis | Cell Import - Endocytosis | Cell Mitochondria | Cell Junctions | Cytoskeleton Introduction | Cytoskeleton - Intermediate Filaments | Cytoskeleton - Microfilaments | Cytoskeleton - Microtubules | Extracellular Matrix 1 | Extracellular Matrix 2 | Cell Cycle | Cell Division | Cell Death 1 | Cell Death 2 | Signal 1 | Signal 2 | Stem Cells 1 | Stem Cells 2 | 2012 Revision | Development


Laboratories: Introduction to Lab | Microscopy Methods | Preparation/Fixation | Immunochemistry | Cell Knockout Methods | Cytoskeleton Exercise | Confocal Microscopy | Microarray Visit | Tissue Culture 1 | Tissue Culture 2 | Stem Cells Lab | Stem Cells Analysis



2012 Projects: Group 1 | Group 2 | Group 3 | Group 4 | Group 5 | Group 6 | Group 7 | Group 8 | Group 9

Dr Mark Hill 2013, UNSW Cell Biology - UNSW CRICOS Provider Code No. 00098G

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