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2011 National Academies Northstar Institute for Undergraduate Education in Biology
Endocytosis: the Dynamic Nature of the Endomembrane
System
Notes to Instructors are italicized on slides and in notes
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Context
1 class session in Introductory Biology for majors
Taught in a large lecture hall
Pre-existing information (from previous lectures)
Cell parts (including cytoskeleton)Membrane structureExocytosis
Reading before class: Textbook section on endocytosis“How Viruses Hijack Endocytic Machinery”
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Goal: Understand the role of endocytosis in cell physiology
Outcomes• Explain steps in endocytosis using the concepts
of molecular recognition and sorting• Explain the function of endocytosis in cell
metabolism• Predict how perturbations of endocytic
processes affects cells• Explain how the endocytic process may be
hijacked by pathogens• Analyze and interpret experimental data• Connect graphical data with visual
representations
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Assessments
• Identify and order the steps of endocytosis• Use concepts of molecular recognition and
sorting to explain specificity in the process• Interpret data about how a perturbation affects
endocytosis and predict consequences for cells• Given information about how a drug affects
endocytosis, analyze experimental data to determine how a pathogen enters a cell
• Predict how drug treatment might prevent a pathogen from reaching the cytosol
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7/14/11
New material is presented in multiple ways including extensive use of animations.
Interactions include everyone in the class.
The unit incorporates a variety of learning activities and student interactions.
Material incorporates diverse examples that will reach different audiences.
Diversity
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Class plan (single class session)
Before class: Students complete assigned reading1. Think-pair-share activity (10 min)
Use a diagram to identify and order steps in the endocytic pathway
2. Lecture (20 min)Key concepts: molecular recognition and
sorting
3. Data analysis and predictions (20 min)Brainstorming and clicker questions
Homework assignment
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How do cells “eat”?
Membrane transporters & channels
Endocytosis
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Pathogens use the endocytic pathway to get into cells
e.g.: flu virus
Salmonella ?
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Think-Pair-Share: Endocytosis
A) THINK INDIVIDUALLY (4 minutes)Read the terms on next slideChoose endocytosis terms Draw a labeled diagram using those
terms
B) PAIR (2 minutes) Discuss your diagram with your neighbor Modify your diagram as needed
C) SHARE (2 minutes) We will use one stellar example in
lectureModify your diagram as we discuss
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Choose appropriate terms and create your own labeled
diagramCargoClathrinCoated pitCoated vesicleEndosomeERGolgiLysosomeMitochondriaNaked vesiclePlasma membraneReceptor
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Lecture starts
Mini-lecture + student input to cover the concepts on the next few slides and videos Remind students to add to their diagram:
Phagocytosis, pinocytosisdifferences and similarities
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Types of Endocytosis
1. Multiple “pathways” of endocytosis2. Molecular recognition between cell
(receptor) and cargo (internalized object)
Biology (2002) Campbell et al.
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Receptor-mediated Endocytosis
Animated Video: Key Points1. Recognition of SPECIFIC cargo2. Multiple steps + compartments3. Consequences for cargo
http://www.youtube.com/watch?v=lNKKg6hIBcM
© Molecular Biology of the Cell (2002) Alberts et al.
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Lecture continues
Cover stepsclathrin
acidification-2 stepsConcepts: Molecular recognition Sorting
cargo-receptorvesicle-vesicle
Why do we need this? © Molecular Biology of the Cell (2002) Alberts et al.
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Phagocytosis
Animated Video: Key Points
1. Similarities to receptor-mediated endocytosis
• Molecular recognition• Sorting
2. Differences• No clathrin
http://www.youtube.com/watch?v=7VQU28itVVw
Animation: © McGraw-Hill
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Phagocytosis
Molecular recognition between cell and bacterium
Lysosomes fuse with phagosome:• Sorting: vesicles specific places• Molecular recognition: lysosomes
phagosomes
What would happen if lysosomes fused with any (all) cellular membranes?
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Exercise:Assess understanding of endocytosis by interpreting real data
A study used a drug that blocks endocytosis to determine how viruses enter/infect cells.
Viruses:Ebola virusVesicular stomatitis virus (VSV)Human Immunodeficiency Virus (HIV)
Drug: Chlorpromazine (Drug C)
Adapted from: Bhattacharyya et al (2010)
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Drug C: effect on normal cell endocytosis
Images show clathrin localization inside cells.
nucleus
nucleus
Clathrin coated pits or vesicles
Clathrin “clumps”
C
Adapted from: Bhattacharyya et al (2010)
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Brainstorm in pairs (2 min):
What steps of endocytosis might be affected by Drug C?
Drug C: effect on normal cell endocytosis
nucleus
nucleus
Clathrin coated pits or vesicles
Clathrin “clumps”
C
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Drug C
Clathrin “clumps”= non-functional
?
?
Adapted from Genes & Dev. 1998. 12: 3663-3674
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% o
f ce
lls in
fect
ed
Drug C
Ebola
Drug C: effect on viral entry into cells
• Test cells received Drug C + virus. • Untreated cells received only
virus.• Control cells received no virus.• 3 viruses: Ebola, VSV and HIV. • Infection is when virus reached
cytosol.
Adapted from: Bhattacharyya et al (2010)
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% o
f ce
lls in
fect
ed
Drug C
Ebola
Drug C: effect on viral entry into cells
• Test cells received Drug C + virus. • Untreated cells received only
virus.• Control cells received no virus.• 3 viruses: Ebola, VSV and HIV. • Infection is when virus reached
cytosol.Clicker Q1 (1 min): Which virus(es) enter the cell by clathrin-mediated endocytosis?
A. Ebola onlyB. VSV onlyC. HIV onlyD. VSV and HIV
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% o
f ce
lls in
fect
ed
Drug C
Ebola
Drug C: effect on viral entry into cells
• Test cells received Drug C + virus. • Untreated cells received only
virus.• Control cells received no virus.• 3 viruses: Ebola, VSV and HIV. • Infection is when virus reached
cytosol.Clicker Q2 (1 min): Which virus(es) require receptors to enter the cell?
A. Ebola onlyB. VSV onlyC. HIV onlyD. Not enough information
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Clicker Q3 (1 min.): Bafilomycin blocks endosome acidification. Which cell diagram represents where Ebola will be located after treatment with this drug? (Small spots = virus.)
A B C D
V CV
CPREDICT THE OUTCOME
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Drug C
Clathrin “clumps”= non-functional
BafilomycinBlocks endosome acidification?
?
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Q1: What is the most likely method by which VSVg enters the cell?
A. Clathrin-mediated endocytosisB. phagocytosisC. pinocytosisD. not enough information
Q2: Justify your answer to Q1
Q3: Based on experiments with Chlorpromazine (discussed in class) and Bafilomycin, what method do you think HIV uses to enter the cell?
Q4: What would happen to a normal cell treated with Bafilomycin, i.e. what are the side effects of this drug?
% o
f ce
lls in
fect
ed
Bafilomycin
This experiment is like the one in class, except cells were treated with Bafilomycin, which inhibits endosome acidification.
HomeworkBafilomycin: effect on viral entry into cells
Ebola