UNSW Cell Biology

2008 ANAT3231 Lecture 06 Cell Importation

Introduction

Objectives

Broad understanding of endocytosis Understanding structure and function of organelles and structures associated with substance import (endocytosis) endosomes lysosomes peroxisomes Golgi apparatus endoplasmic reticulum Brief understanding of phagocytosis Brief understanding of viral and bacterial entry into the cell

Lecture Audio

The University has a system for automated recording of lectures called Lectopia (or iLecture).

Lectopia recording: 2008 ANAT3231 Lecture 6 - Cell Import

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Links: Lectopia Login Page | Cell Biology Podcast Page | Current Course Outline 2008

History

Links: 1974 Nobel Prize |

1974 Nobel Prize

Links: 1974 Nobel Prize | UCSD - Palade Lab |

Cell Fractionation Techniques

Links: MBOC - Cell fractionation by centrifugation |

Cytosolic Vesicles

Single membrane bound vesicles two membrane processes involved in trafficking budding fusion Linked to the ER and Golgi membrane system Lysosomes Peroxisomes

Peroxisomes

increased activity of digestion in enzyme studies de Duve C. The peroxisome: a new cytoplasmic organelle. Proc R Soc Lond B Biol Sci. 1969 Apr 15;173(30):71-83. http://www.ncbi.nlm.nih.gov/pubmed/4389648 Identified by EM 10 years later enzymes that produce and others that degrade hydrogen peroxide (a reactive oxygen species, ROS) oxidative reactions using molecular oxygen to generate hydrogen peroxide oxidizing fatty acids, bile salts and cholesterol then converting hydrogen peroxide to nontoxic forms Catalases (EC 1.11.1.6) haem-containing proteins that catalyse conversion of hydrogen peroxide (H2O2) to water and molecular oxygen

Links: MBOC - EM Peroxisomes | MBOC - EM Plant Peroxisomes | MBOC - Peroxisomes | The Cell - Peroxisomes | Lippincott-Schwartz Lab | Electron micrograph of rat liver | MCB - synthesis of catalase | interpro - catalase |

Peroxisome Assembly

• Two theories on peroxisome formation
• semiautonomous oranelles (like mitochondria) which multiply strictly by growth and division
  • Free ribosomes synthesize peroxisome proteins
  • Imported into pre-existing peroxisomes as completed polypeptide chains
  • Peroxisome growth from protein import
  • formation of new peroxisomes by division of old ones
• other organelles such as ER role in formation and maintenance of peroxisomal membranes

Links: MBOC - A model for how new peroxisomes are produced | Lippincott-Schwartz Lab |

Absorption Mechanisms

• Endocytosis receptor mediated endocytosis
  • General term for all mechanisms of absorbtion extracellular fluid and substances
  • substances bind to receptor sites
  • vesicle called endosome
• Pinocytosis
  • "cell drinking"
  • All cells, extracellular fluid
  • micropinocytosis within vesicles (<0.1 µm diameter)
  • macropinocytosis within vacuoles (0.5-5.0 µm) named macropinosomes
  • 
  • From: Protein Helps Orchestrate Cells' Fluid Uptake PLoS Biology Vol. 2, No. 9, e318 doi:10.1371/journal.pbio.0020318
• Phagocytosis
  • “cell eating”
  • occurs only in specialized cells macrophages, dendritic cells and neutrophils
  • capture and destroy pathogens and particulate antigens
  • essential component of the innate immune response
  • Fc- and complement-receptor mediated phagocytosis
    • named for binding specificity for antibody tail region called Fc (Fragment, crystallizable)
  • clearance of apoptotic bodies
  • (MH - some bacteria "hijack" this process in non-phagocytic cells to enter and infect them)

Links: NRG - The role of the endosomal–lysosomal apparatus | MBOC - Mechanisms used by bacteria to induce phagocytosis by nonphagocytic host cells |

Phagocytosis Movie

Remember the white blood cell chasing and phagocytosing the bacteria in a blood smear.

About the Movie: This video is taken from a 16mm movie made in the 1950s by the late David Rogers at Vanderbilt University. It was given to Dr. Tom Stossel via Dr. Viktor Najjar, Professor Emeritus at Tufts University Medical School and a former colleague of Rogers. It depicts a human polymorphonuclear leukocyte (neutrophil) on a blood film, crawling among red blood cells, notable for their dark color and principally spherical shape. The neutrophil is "chasing" Staphylococcus aureus microorganisms, added to the film. (Text from- Tom Stossel, June 22, 1999)

Links: MBOC - Phagocytosis |

Endosome

vesicle formed from plasma membrane budding encloses extracellular fluid and substances large ones called a phagosome or vacuole

Links: MBOC - Image - The endocytic pathway from the plasma membrane to lysosome | MBOC - Image - Lysosomes

Autophagy

lysosomal degradation pathway for cytoplasmic material survival mechanism during short-term starvation

(A) Upon induction of autophagy, a membrane sac called the isolation membrane (IM) forms and engulfs portions of the cytoplasm. Sealing of its edges gives rise to the double-membrane bound autophagosome. Fusion of the outer membrane with a lysosome results in formation of an autolysosome, in which the inner autophagosomal membrane and its contents are degraded. From: PLOS - Autophagy: A Forty-Year Search for a Missing Membrane Source | Juhasz G, Neufeld TP PLoS Biology Vol. 4, No. 2, e36 doi:10.1371/journal.pbio.0040036

Links: MBOC - Image - Three pathways to degradation in lysosomes | eurekah - Image - EM Morphology of autophagosomes and autophagic vacuoles in isolated mouse hepatocytes | PLOS - Autophagy: A Forty-Year Search for a Missing Membrane Source |

Lysosomes

• Are the site of cellular digestion
  • contain up to 40 enzymes for digestion
• Acid Hydrolases
  • Active at acid pH (5)
• Hydrogen ion pump in lysosomal membrane
  • drives ions from cytoplasm into lumenal space
  • generates internal acidic environment

Lysosome Membrane

• Allows passage of uncharged molecules
• Molecules enter, are charged and cannot leave

Lysosome Digestive Enzymes

• Acid hydrolases
• enzymes named on basis of substrate
  • Protease - digests proteins
  • Nuclease - digests neucleotides (DNA)
  • Glycosidase - digests carbohydrates (sugars)
  • Lipases - digests lipids (fats)
  • Phospholipases - digests phospholipids (membranes)
  • Phosphatases - removes a phosphate group

Lysosome Types

• Primary
  • newly formed without digestive substrate
  • formed from budding Golgi apparatus
  • can be secreted by exocytosis
• Secondary
  • active form enzyme + substrate
  • formed by vesicle fusion event

(MH - primary lysosomes in neutrophils are called primary granules or A granules)

Lysosome-Associated Membrane Proteins

• LAMP-1, LAMP-2
• Knockout of LAMP-2 in mice lysosomal enzyme targeting, autophagy and lysosomal biogenesis

Caveolae

small membrane invaginations defined by containing caveolin protein in the vesicle membrane not always present in all cells functions lipid recycling cellular signalling endocytosis

Main features of caveolae and caveolins. The electron micrographs in panels a and b show caveolae in adipocytes that have been surface-labelled with an electron-dense marker. Panel c shows a glancing section across the cell surface of a primary fibroblast that has been similarly labelled. Caveolae are evident as discrete flask-shaped pits, or circular profiles where the surface connection lies outside the plane of the section. Note the complex forms of surface-connected caveolae in the adipocytes (panels a and b), and the incredible abundance of caveolae in specific regions of the fibroblast surface (panel c). Panel d indicates how caveolin is inserted into the caveolar membrane, with the N and C termini facing the cytoplasm and a putative 'hairpin' intramembrane domain embedded within the membrane bilayer. The scaffolding domain, a highly conserved region of caveolin, might have a role in cholesterol interactions through conserved basic (+) and bulky hydrophobic residues (red circles). The C-terminal domain, which is close to the intramembrane domain, is modified by palmitoyl groups that insert into the lipid bilayer. The complex structures that are formed by interconnected caveolae can occupy a large area of the plasma membrane. The hypothetical formation of cubic membranes (panel e), which have adapted to allow the invagination of numerous caveolae, is depicted schematicall. These membrane invaginations can form with little energy input. (Image: Parton RG, Simons K. The multiple faces of caveolae. Nat Rev Mol Cell Biol. 2007 Mar;8(3):185-94. Review.PMID: 17318224 )

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 microtubule-based transport

Endoplasmic Reticulum and Golgi

• Both these systems involved with both cell import and export
• The question is how these compartments "sort" components going in different directions?
• By this stage you should now be able to fully label the figure below.

Other Vesicles

• Exosomes
  • nanometer-sized membrane vesicles invaginating from multivesicular bodies and secreted from different cell types
  • Function suggested as the eradication of obsolete proteins, antigen presentation, or "Trojan horses" for viruses or prions. (PMID: 16809645)
• endosomal multivesicular bodies (MVBs)/endosomes
  • a stage in endosomal development
  • compartments for receptor downregulation and as intermediates in the formation of secretory lysosomes. (PMID: 12892785)
  • delivery of transmembrane proteins into the lumen of the lysosome for degradation is mediated by the multivesicular body pathway. (PMID: 15569240)
  • MVB formation occurs when a portion of the limiting membrane of an endosome invaginates and buds into its own lumen. Intralumenal vesicles are degraded when MVBs fuse to lysosomes. (PMID: 17506697)
  • The ESCRT (ESCRT-I, -II and -III) complexes form a network that recruits monoubiquitinated proteins and drives their internalization into lumenal vesicles within a type of endosome known as a multivesicular body. (PMID: 16689637)
  • essential for both sorting and multivesicular endosomes formation (PMID: 12892785)
• Synaptic vesicles
  • neuron specific
  • filled with neurotransmitter
• Macropinosomes
  • Macropinocytosis defines a series of events initiated by extensive plasma membrane reorganization or ruffling to form an external macropinocytic structure that is then enclosed and internalized. The process is constitutive in some organisms and cell types but in others it is only pronounced after growth factor stimulation. Internalized macropinosomes share many features with phagosomes and both are distinguished from other forms of pinocytic vesicles by their large size, morphological heterogeneity and lack of coat structures. (PMID: 17760832)
• Phagosomes fusion of endoplasmic reticulum (ER) with macrophage plasmalemma, underneath phagocytic cups, is a source of membrane for phagosome formation in macrophages (PMID: 12151002)
• phagocytic cup
  • actin-based membrane structure formed at the plasma membranes
  • impaired in Wiskott-Aldrich syndrome (WAS)
• Melanosomes

Abnormalities

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

References

• Essential Cell Biology - Chapter 7
• Molecular Biology of the Cell (4th ed.) - Part 1 Chapter 8 - The Cell Nucleus
  • Alberts, Bruce; Johnson, Alexander; Lewis, Julian; Raff, Martin; Roberts, Keith; Walter, Peter New York and London: Garland Science; c2002

NLM Online Textbooks

NCBI Bookshelf

• Molecular Biology of the Cell 4th ed. - Part 1 Chapter 12
  • Alberts, Bruce; Johnson, Alexander; Lewis, Julian; Raff, Martin; Roberts, Keith; Walter, Peter New York and London: Garland Science; c2002
• The Cell - A Molecular Approach - Chapter 9. Protein Sorting and Transport - The Endoplasmic Reticulum, Golgi Apparatus, and Lysosomes
  • Cooper, Geoffrey M. Sunderland (MA): Sinauer Associates, Inc. ; c2000
• Molecular Cell Biology - Chapter 17. Protein Sorting: Organelle Biogenesis and Protein Secretion
  • Lodish, Harvey; Berk, Arnold; Zipursky, S. Lawrence; Matsudaira, Paul; Baltimore, David; Darnell, James E. New York: W. H. Freeman & Co. ; c1999

Web Links

Molecular Biology of the Cell The Endoplasmic Reticulum

Intracellular Compartments and Protein Sorting

Transport from the ER through the Golgi Apparatus

Vesicular Traffic

Golgi Apparatus- Summary

Cell Biology Topics- University of Arkansas for Medical Sciences

Nucleus

Role of the Ribosome

Rough Endoplasmic Reticulum

Smooth Endoplasmic Reticulum

Golgi Complex: Structure and Function

Web Movies

Molecular Cell Biology

First link is directly to Quicktime move, followed by associated textbook figure with legend. Look at the movie first, then look at the labelled image.

MCB - Protein Secretion | Figure 17-13. The secretory pathway of protein synthesis and sorting

JCB - organelles and vesicles display bidirectional motility along microtubules | JCB - nocodazole disperses organelles away from the perinuclear region and also slows vesicle motility

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Acronyms

• AA - amino acid
• DNA - deoxyribonucleic acid
• EM - electron microscopy
• Fc- Fragment, crystallizable region of antibody
• FL - flourescent
• LAMP - Lysosome-Associated Membrane Protein
• MPS - Mucopolysaccharidosis
• MVB - endosomal MultiVesicular Bodies
• OMIM - Online Mendelian Inheritance in Man Database
• RER - rough endoplasmic reticulum
• RNA - ribonucleic acid
• ROS - reactive oxygen species
• SER - smooth endoplasmic reticulum
• WAS - Wiskott-Aldrich Syndrome

2007 Lecture Slides

(MH - note that content will not match exactly current lecture structure but has been selected as having similar content)

lecture04 1 slide/page (view only) (53 pages, 1 Mb)

lecture04 6 slides/page (print) (9 pages, 516 Kb)

lecture04 outline (print no images) (6 pages, 92 Kb)

See also Lecture Slides Text on this current page

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Links: Download Acrobat Reader 8.0

Comments

The topic of cell importation is very current as research is continuing to establish mechanisms involved with vesicle trafficking and transport. There are many new terms and mixed nomenclature, so spend some time to get a broad overview of the topic before going into too much detail. An important concept to understand is the formation of many different structural compartments within the cell by a simple lipid membrane, which then allows functional specialization.

Also realise that some processes are cell type and cell cycle specific, for example with phagocytosis, with others being more general, endocytosis.

	In 2008 a new way of presenting course content online is being trialled. Please let me know of any difficulties/suggestions or things that work well. Notice also that in some slides I have added annotations in brackets with my initials (MH - )
	Links: Current Course Outline 2008