2009 Lecture 5

From CellBiology

Cell Export - Exocytosis

Exocytosis and Endocytosis cartoon

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).

Too read this current page requires signal transmission between neurons, by synaptic vesicle release, a form of exocytosis.

Homework - What did you find difficult to understand about exocytosis? 2009 Students Page

Dr Mark Hill (2009)

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. 2009 All Audio Files

Lecture 5: Cell Export - Exocytosis Lecture Date: 24-03-2009 Lecture Time: 13:00 Venue: BioMed F Speaker: Mark Hill

Archive

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

Below are some example historical research finding related to cell junctions 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.


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. | MCB - movie - Protein Secretion

The Cytosol

  • 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: MCB - Figure 17-1. Overview of sorting of nuclear-encoded proteins in eukaryotic cells

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

Transfer RNA (tRNA)

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

Ribosomal RNA (rRNA)

  • 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

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

Ribosomes

Ribosome Structure

  • 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

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

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 | MCB - movie ch4anim4.mov

Endoplasmic Reticulum

* 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)
Nucleus and RER tem
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
  • no attached ribosomes
  • not involved in protein synthesis
  • differ in shape
  • SER a meshwork of fine tubules

Smooth Endoplasmic Reticulum - Function

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

(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

* 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
    • 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


Tem golgi1.jpg
Tem golgi2.jpg

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 complex built by analyzing micrographs of serial sections through a secretory cell

Golgi Apparatus - Functions

  • 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


Links:

Secretory Vesicles

  • protein export (secretion)
    • constitutive and regulated
  • related also to membrane turnover
    • new lipid
    • new cholesterol
    • new membrane proteins


Exocytosis types
Docked secretory vesicles
Exo endo cytosis.jpg Movies

JCB - movie - transport vesicles and lipid (large 9.7 Mb)

JCB - movie - View of many Carriers

"There are three types of behaviors of the post-Golgi carriers: (1) carriers that are stationary; (2) carriers that are moving, but not fusing; and (3) carriers that fuse to the plasma membrane. As can be seen on the extreme left and right, fusion occurs with a bright local burst of fluorescence followed by a spread of the fluorescence. The carrier on the extreme left was observed doing transport, docking, and fusion during the acquisition time. The two carriers on the extreme right did not move significantly before they fused."

Links: JCB - movie - View of many Carriers (2.2 Mb) | 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

  • Molecular Cell Biology - Chapter 17. Protein Sorting: Organelle Biogenesis and Protein Secretion
  • The Dynamic Cell

The Cell- A Molecular Approach

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

Search Online Textbooks

Books

PubMed

  • PubMed is a service of the U.S. National Library of Medicine that includes over 18 million citations from MEDLINE and other life science journals for biomedical articles back to 1948. PubMed includes links to full text articles and other related resources. PubMed
  • PubMed Central (PMC) is a free digital archive of biomedical and life sciences journal literature at the U.S. National Institutes of Health (NIH) in the National Library of Medicine (NLM) allowing all users free access to the material in PubMed Central. PMC
  • 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

Search Pubmed

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.


Working area

  • exocytosis - historically called "emiocytosis"
  • endocytosis - phagocytosis for solids, pinocytosis for fluids
  • SNARE complex formed from SNARE proteins (Soluble N-ethylmaleimide sensitive factor Attachment protein REceptor) the role is to mediate fusion of cellular transport vesicles with the cell membrane (exocytosis) or with a target compartment such as a lysosome (endocytosis). PMID: 8221884
  • trans golgi network - face of the trans-Golgi network from which vesicles leave the Golgi apparatus.
  • Terms: endocytic TGN, Golgi apparatus, multivesicular bodies (MVBs), ribosomes, secretory vesicle,
  • Search: SNARE protein | multivesicular bodies
  • regulated secretory pathway in neuroendocrine cells
  • Lamellar bodies are lysosome-related secretory granules and store lung surfactant in alveolar type II cells.
  • Plants desmotubule - derived from smooth endoplasmic reticulum passing through the plasmodesmata.
header 1 header 2 header 3
Ribosomes_bound_mRNA_cartoon1
Mammalian proteins transported into er
Export from the ER in living cells
Post-Golgi_transport_cartoon
Golgi apparatus endocytic TGN
row 2, cell 3

Links

Homework - What did you find difficult to understand about exocytosis?


2009 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 1 Intermediate Filaments | Cytoskeleton 2 Microtubules | Cytoskeleton 3 Microfilaments | Extracellular Matrix 1 | Extracellular Matrix 2 | Cell Cycle | Cell Division | Cell Death 1 | Cell Death 2 | Signal 1 | Signal 2 | Stem Cells | Stem Cells | Development | Revision

Laboratories

Introduction to Lab | Microscopy Methods | Preparation/Fixation | Immunochemistry | Cell Knockout Methods | Cytoskeleton Exercise | Confocal Microscopy | Tissue Culture 1 | Tissue Culture 2 | Microarray Lab visit

Dr Mark Hill 2015, UNSW Cell Biology - UNSW CRICOS Provider Code No. 00098G