Difference between revisions of "Cell Import - Endocytosis"
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Please note the lecture will be combined with exocytosis on Thursday.
Please note the lecture will be combined with exocytosis on Thursday.
Latest revision as of 11:03, 12 April 2017
- 1 Cell Import - Endocytosis
- 2 Objectives
- 3 Cytosolic Vesicles
- 4 Peroxisomes
- 5 Absorption Mechanisms
- 6 Endosome
- 7 Autophagy
- 8 Lysosomes
- 9 Proteasomes
- 10 Caveolae
- 11 Transport Vesicles
- 12 Endoplasmic Reticulum and Golgi
- 13 Other Vesicles
- 14 Abnormalities
- 15 History
- 16 References
- 17 Images
- 18 Movies
- 19 2017 Course Content
Cell Import - Endocytosis
Associate Professor Till Böcking leads the Molecular Machines Group in the EMBL Australia Node for Single Molecule Science.
His slides will be added to this page when available.
Lecture Slides: 2017 Lecture PDF
Please note the lecture will be combined with exocytosis on Thursday. Here is an earlier archive recording of Endocytosis Lecture
The content below is for information purposes only and contains links to the online textbooks. This will not be used for the lecture presentation.
We will study this topic at the level of the cellular components and organelles involved in the process: endosomes, lysosomes, peroxisomes, transport vesicles, Golgi apparatus and endoplasmic reticulum.
- Understanding of the process of endocytosis
- Understanding structure and function of organelles and structures associated with substance import (endocytosis) including:
- endosomes, lysosomes, peroxisomes, Golgi apparatus and endoplasmic reticulum
- Understanding of other cell import mechanisms, phagocytosis etc.
- Brief understanding of signal, viral and bacterial entry into the cell
- Brief understanding of transport within the cell
|Visualization of Rab9-mediated vesicle transport from endosomes to the trans-Golgi in living cells. Barbero P, Bittova L, Pfeffer SR. J Cell Biol. 2002 Feb 4;156(3):511-8. Epub 2002 Feb 4. PMID 11827983 GFP-Rab9-bearing organelles and vesicles display bidirectional motility along microtubules. Speed is 5 times actual.|
- Single membrane bound vesicles
- two membrane processes involved in trafficking
- Linked to the ER and Golgi membrane system
- organelles that metabolize fatty acids
- 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. PMID 4389648 (originally called microbodies)
- 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 220.127.116.11)
- haem-containing proteins that catalyse conversion of hydrogen peroxide (H2O2) to water and molecular oxygen
- Links: MBOC - EM Peroxisomes | MBOC - Peroxisomes | The Cell - Peroxisomes | Lippincott-Schwartz Lab | Electron micrograph of rat liver | MCB - synthesis of catalase
- 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
Receptor Mediated Endocytosis
- General term for all mechanisms of absorbtion extracellular fluid and substances
- substances bind to receptor sites
- vesicle called endosome
- can be utilized by viruses to enter cells
- "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
- “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
- some bacteria "hijack" this process in non-phagocytic cells to enter and infect them
Neutrophil chasing Bacteria and Phagocytosis
|Remember this movie from the first lecture, where the white blood cell chases and phagocytoses a 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)
- vesicle formed from plasma membrane budding
- encloses extracellular fluid and substances
- large ones called a phagosome or vacuole
- pH 5.5 - recycling endosomes and intracellular secretory vesicles PMID 25385186
- Clathrin is a protein that coats both small membrane pits and coated vesicles
- formed during endocytosis of materials at the surface of cells
- macromolecules to be internalized bind to specific cell surface receptors
- receptors are concentrated in regions of the plasma membrane (clathrin-coated pits)
- pits bud from the membrane to form small clathrin-coated vesicles containing the receptors and bound macromolecules
- lysosomal degradation pathway for cytoplasmic material
- survival mechanism during short-term starvation
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
- 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
- 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
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
The ubiquitin proteasome system is an important non-lysosomal protein degradation pathway.
- a protein complex that degrades proteins by proteolysis
- misfolded, unneeded or damaged proteins
- not vesicles, proteins form a "stacked-ring" structure
- proteins destined for destruction are ubiquitinated (ubiquitination)
- attachment of one or more ubiquitin monomers to protein
- small membrane invaginations
- defined by containing caveolin protein in the vesicle membrane
- not always present in all cells
- lipid recycling
- cellular signalling
Links: Parton RG, Simons K. The multiple faces of caveolae. Nat Rev Mol Cell Biol. 2007 Mar;8(3):185-94. Review. PMID: 17318224 FIGURE 3 Caveola endocytosis | Madame Curie Bioscience Database - Transmission electron micrographs of endothelial caveolae
- 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
- also may use microfilament transport
Endoplasmic Reticulum and Golgi
- Both these systems involved with both cell import and export
- New proteins synthesized on membrane-bound ribosomes are transported through the Golgi apparatus
- reach the trans-Golgi network (TGN) and sorted for delivery to various destinations
- exocytosis and endocytosis pathway
- The question is how these compartments "sort" components going in different directions?
- biodigested products from the digestive lysosomal pathway need now to be delivered to the biosynthetic pathway
- amino acids, nucleotides, carbohydrates, phospholipids, lipids, etc
By this stage you should now be able to fully label this figure
- 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
- A type of cytoplasmic vesicle (200 - 500 nmdiameter) that occurs when part of an endosome membrane invaginates and buds into its own lumen forming smaller contained vesicles.
- smaller contained vesicles are degraded when the endosome fuses with a lysosome.
- allows delivery of transmembrane proteins into the lumen of the lysosome for degradation.
- 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)
- 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)
Links: Biogenesis and function of multivesicular bodies. Piper RC, Katzmann DJ. Annu Rev Cell Dev Biol. 2007;23:519-47. Review. PMID: 17506697
- 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)
- 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)
- secreted vesicle
- neuron specific
- filled with neurotransmitter
- membrane vesicle enclosing melanin
- melanin is a light-absorbing pigment
- skin melanocytes and retinal pigment epithelium cells
- melanophages are cells that engulfed the released melanosomes (eg skin keratinocytes)
- skin colour due to melanocytes level of activity not to the number of melanocytes
- Several inherited disorders of lysosomal metabolism
- tested at birth by Gutherie heel-prick
- OMIM Database 112 entries
- Lack of a specific enzyme
- Can isolate and measure enzyme activities
- LAMP-2 deficiency in humans
- fatal cardiomyopathy and myopathy
- OMIM http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=300257
Mucopolysaccharidosis (MPS) IIIB (Sanfilippo Syndrome type B)
- children develop disturbances of sleep, activity levels, coordination, vision, hearing, and mental functioning culminating in early death
- deficiency in lysosomal enzyme N-acetyl-glucosaminidase (Naglu)
Peroxisome biogenesis disorders
(PBDs) Zellweger syndrome and neonatal adrenoleukodystrophy are fatal genetic diseases that are autosomal recessive.
Genes and Diseases
- Genes and Disease selected range of human disorders by system.
- OMIM Online Mendelian Inheritance in Man, an online database of genetic disorders and genes.
Below are some example historical research finding related to endocytosis from the JCB Archive.
1956 Catching sight of lysosomes Lysosomes are identified by Christian deDuve when a membrane barrier gradually dissolves, thus yielding the tell-tale release of an enzyme activity over time.
1964 Coated pits bring in the yolk A study of yolk protein uptake leads Thomas Roth and Keith Porter to propose that endocytosis is specific to a particular cargo and that the vesicle coat might be functioning in both selection and mechanical molding.
1967 How to make a lysosome Daniel Friend and Marilyn Farquhar find that transport pathways intersect: synthesized enzyme meets endocytosed protein in the lysosome.
1978 Viruses catch an endocytic ride into the cell Ari Helenius puts together snapshots of virus entry to form a coherent sequence of events.
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.
pHuji, a pH-sensitive red fluorescent protein for imaging of exo- and endocytosis
J Cell Biol. 2014 Nov 10;207(3):419-32. doi: 10.1083/jcb.201404107.
Shen Y1, Rosendale M2, Campbell RE3, Perrais D4.
Fluorescent proteins with pH-sensitive fluorescence are valuable tools for the imaging of exocytosis and endocytosis. The Aequorea green fluorescent protein mutant superecliptic pHluorin (SEP) is particularly well suited to these applications. Here we describe pHuji, a red fluorescent protein with a pH sensitivity that approaches that of SEP, making it amenable for detection of single exocytosis and endocytosis events. To demonstrate the utility of the pHuji plus SEP pair, we perform simultaneous two-color imaging of clathrin-mediated internalization of both the transferrin receptor and the β2 adrenergic receptor. These experiments reveal that the two receptors are differentially sorted at the time of endocytic vesicle formation. © 2014 Shen et al.
Regulation of peroxisome dynamics
Curr Opin Cell Biol. 2009 Feb;21(1):119-26. doi: 10.1016/j.ceb.2009.01.009. Epub 2009 Jan 31.
Smith JJ, Aitchison JD. Source Institute for Systems Biology, 1441 N 34th Street, Seattle, WA 98103, USA. firstname.lastname@example.org
Peroxisomes are single-membraned organelles ubiquitous to eukaryotic cells that house metabolic reactions that generate and destroy harmful oxidative intermediates. They are dynamic structures whose morphology, abundance, composition, and function depend on the cell type and environment. Perhaps due to the potentially damaging and protective metabolic roles of peroxisomes and their dynamic presence in the cell, peroxisome biogenesis is emerging as a process that involves complex underlying mechanisms of regulated formation and maintenance. There are roughly 30 known peroxins, proteins involved in peroxisome biogenesis, many of which have been conserved from yeast to mammals. This review focuses on the biogenesis of peroxisomes with an emphasis on the regulation of peroxisome formation and the import of peroxisomal matrix proteins in the model organism Saccharomyces cerevisiae. PMID 19188056
Clathrin couture: fashioning distinctive membrane coats at the cell surface
PLoS Biol. 2009 Sep;7(9):e1000192. Epub 2009 Sep 8.
Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA. email@example.com Comment on:
PLoS Biol. 2009 Sep;7(9):e1000191. PMID 19809570 http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1000192
Membrane organization and dynamics in cell polarity
Cold Spring Harb Perspect Biol. 2009 Nov;1(5):a001321.
Orlando K, Guo W.
Department of Biology, University of Pennsylvania, Philadelphia, 19104-6018, USA. Abstract The establishment and maintenance of cell polarity is important to a wide range of biological processes ranging from chemotaxis to embryogenesis. An essential feature of cell polarity is the asymmetric organization of proteins and lipids in the plasma membrane. In this article, we discuss how polarity regulators such as small GTP-binding proteins and phospholipids spatially and kinetically control vesicular trafficking and membrane organization. Conversely, we discuss how membrane trafficking contributes to cell polarization through delivery of polarity determinants and regulators to the plasma membrane.
Essential Cell Biology
- Chapter 14 Intracellular Compartments and Transport, Endocytic Pathways p472
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 Biology of the Cell 4th ed. - Part 1 Chapter 12
- Figure 13-57. Figure 13-1. The endocytic and biosynthetic-secretory pathways
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
- 17.9. Receptor-Mediated Endocytosis and the Sorting of Internalized Proteins
- Lysosomes Are Acidic Organelles That Contain a Battery of Degradative Enzymes
The Cell- A Molecular Approach
Cooper, Geoffrey M. Sunderland (MA): Sinauer Associates, Inc.; c2000
- The Cell - A Molecular Approach - III. Cell Structure and Function Chapter 12 The Cell Surface
- Endocytosis and Lysosome Formation
Search Online Textbooks
- "endocytosis" Molecular Biology of the Cell | Molecular Cell Biology | The Cell- A molecular Approach | Bookshelf
- "lysosome" Molecular Biology of the Cell | Molecular Cell Biology | The Cell- A molecular Approach | Bookshelf
- "autophagy" Molecular Biology of the Cell | Molecular Cell Biology | The Cell- A molecular Approach | Bookshelf
- 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
- "endocytosis" PubMed reviews | PubMed all articles | PMC reviews | PMC all articles | OMIM | Entrez all databases
- "receptor-mediated endocytosis" PubMed reviews | PubMed all articles | PMC reviews | PMC all articles | OMIM | Entrez all databases
- "clathrin" PubMed reviews | PubMed all articles | PMC reviews | PMC all articles | OMIM | Entrez all databases
- "lysosome" PubMed reviews | PubMed all articles | PMC reviews | PMC all articles | OMIM | Entrez all databases
- "autophagy" PubMed reviews | PubMed all articles | PMC reviews | PMC all articles | OMIM | Entrez all databases
- The endocytic matrix. Scita G, Di Fiore PP. Nature. 2010 Jan 28;463(7280):464-73. Review. PMID: 20110990
- Endocytosis and signalling: intertwining molecular networks. Sorkin A, von Zastrow M. Nat Rev Mol Cell Biol. 2009 Sep;10(9):609-22. Review. PMID: 19696798
- PMID: 15030567
- Endosome to trans-Golgi traffic is vesicular Coexpression of fluorescent Rab9 and Rab7 by Barbero et al. revealed that these two late endosome Rabs occupy distinct domains within late endosome membranes. Rab9 is present on endosomes that display bidirectional microtubule-dependent motility, and Rab9-positive transport vesicles (rather than tubules) can be seen moving along microtubule tracks and fusing with the trans-Golgi network.
- Recycling of endosomes by Arf6 Activation of nucleotide exchange on Arf6 causes an increase in both membrane internalization and the return of the resultant structures to the plasma membrane. However, when Brown et al. block Arf6 in its GTP-loaded ("on") state, this results in accumulation of endosome-derived vacuoles.
- Actin tails form behind motile endosomes and lysosomes Taunton et al. document the formation of actin comet tails (red) behind motile endosomes and lysosomes. The tails may help the organelles move around the cell, and influence the construction of the cytoskeleton.
- Lysosomes repair the plasma membrane When cells are injured, calcium rushes in and prompts exocytosis of membranes to repair the gap. Jaiswal et al. confirm that the repairing membranes are from lysosomes.
- Vam6p clusters and fuses lysosomes Caplan et al. find that human Vam6p promotes clustering and then fusion of lysosomes.
2017 Course Content
Lectures: Cell Biology Introduction | Cells Eukaryotes and Prokaryotes | Cell Membranes and Compartments | Cell Nucleus | Cell Export - Exocytosis | Cell Import - Endocytosis | Cytoskeleton Introduction | Cytoskeleton - Microfilaments | Cytoskeleton - Microtubules | Cytoskeleton - Intermediate Filaments | Cell Mitochondria | Cell Junctions | 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 | Development | 2017 Revision
Dr Mark Hill 2015, UNSW Cell Biology - UNSW CRICOS Provider Code No. 00098G