2009 Lecture 6

From CellBiology
Exocytosis and Endocytosis cartoon

Cell Import - Endocytosis

Endocytosis types

This lecture introduces how substances that are imported (endocytosis) and processed by the cell.

Notes in Preparation (notice removed when complete)

There are a number of processes by which the cell can absorb substances. The two main forms are by endocytosis or phagocytosis. Absorbed substances include: substances required for cell growth, cell signaling toxic chemicals and drugs, bacteria and viruses. In addition, this is the main method for membrane removal and recycling.

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.

Movie Vesicles Display Bidirectional Motility along Microtubules 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.

Objectives

  • 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

Lecture Audio

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

History - 1974 Nobel Prize

  • Albert Claude
    • electron microscope for the study of animal cells and for development of differential centrifugation
  • George Palade
    • methodological improvements both differential centrifugation and electron microscopy
    • discovered and described small granular components now known as ribosomes
  • Christian de Duve
    • identification of the isolated components which were named lysosomes

Links: 1974 Nobel Prize | UCSD - Palade Lab |

Cell Fractionation Techniques

  • Centrifugation generated 4 fractions
    • Nuclear
    • Mitochondrial
    • Microsomal
    • Supernatant

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

Membrane compartments

TEM Golgi Apparatus

Peroxisomes

Peroxisome
  • increased activity of digestion in enzyme studies
  • 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 Types

Endocytosis Mechanisms

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

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
  • 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 |

Neutrophil chasing Bacteria and Phagocytosis

Phagocytosis Movie

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)

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

Links: Lipid rafts and membrane dynamics


Clathrin-coated Pits

Clathrin-coated Pits

  • Clathrin is a protein that coats both small membrane pits and coated vesicles
    • formed during endocytosis of materials at the surface of cells

Autophagy

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

Autophagy

(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


autophagy types

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

Lysosomes primary and secondary

  • 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
  • primary lysosomes in neutrophils are called primary granules or A granules

Links: ASCB - Lysosome History Series: 5. Historic Examples of Primary Lysosomes | NRG - The role of the endosomal - lysosomal apparatus

Lysosome-Associated Membrane Proteins

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

Caveolae

Caveolae myoepithelial cells 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

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
    • also may use microfilament transport

Endoplasmic Reticulum and Golgi

Endocytic Transgolgi Network (TGN)
  • 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

By this stage you should now be able to fully label this figure

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
  • 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

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)

Synaptic Vesicles

  • secreted vesicle
  • neuron specific
  • filled with neurotransmitter

Melanosomes

  • 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


Proteasomes

  • 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

Abnormalities

Lysosomal Disorders

  • 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

Danon disease

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)

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.

History

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.

References

Textbooks

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 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

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

Articles

Working Area

  • clathrin-independent endocytic pathways
  • Autophagy is activated for cell survival after endoplasmic reticulum stress. Ogata M, Hino S, Saito A, Morikawa K, Kondo S, Kanemoto S, Murakami T, Taniguchi M, Tanii I, Yoshinaga K, Shiosaka S, Hammarback JA, Urano F, Imaizumi K. Mol Cell Biol. 2006 Dec;26(24):9220-31. Epub 2006 Oct 9. PMID: 17030611
    • "Eukaryotic cells deal with accumulation of unfolded proteins in the endoplasmic reticulum (ER) by the unfolded protein response, involving the induction of molecular chaperones, translational attenuation, and ER-associated degradation, to prevent cell death. Here, we found that the autophagy system is activated as a novel signaling pathway in response to ER stress. Treatment of SK-N-SH neuroblastoma cells with ER stressors markedly induced the formation of autophagosomes, which were recognized at the ultrastructural level. The formation of green fluorescent protein (GFP)-LC3-labeled structures (GFP-LC3 "dots"), representing autophagosomes, was extensively induced in cells exposed to ER stress with conversion from LC3-I to LC3-II. In IRE1-deficient cells or cells treated with c-Jun N-terminal kinase (JNK) inhibitor, the autophagy induced by ER stress was inhibited, indicating that the IRE1-JNK pathway is required for autophagy activation after ER stress. In contrast, PERK-deficient cells and ATF6 knockdown cells showed that autophagy was induced after ER stress in a manner similar to the wild-type cells. Disturbance of autophagy rendered cells vulnerable to ER stress, suggesting that autophagy plays important roles in cell survival after ER stress."
  • Abcam - Receptor-mediated endocytosis by clathrin-coated vesicles
  • Virus can enter the cell by receptor-mediated endocytosis. Acidification of the endocome, breaks dow the virus capsid.
    • For example: adenovirus virions bind to the coxsackie adenovirus receptor (CAR) and integrins on the plasma membrane.

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