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

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”

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

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

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

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

How do cells “eat”?

Membrane transporters & channels

Endocytosis

Pathogens use the endocytic pathway to get into cells

e.g.: flu virus

Salmonella ?

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

Choose appropriate terms and create your own labeled

diagramCargoClathrinCoated pitCoated vesicleEndosomeERGolgiLysosomeMitochondriaNaked vesiclePlasma membraneReceptor

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

Types of Endocytosis

1. Multiple “pathways” of endocytosis2. Molecular recognition between cell

(receptor) and cargo (internalized object)

Biology (2002) Campbell et al.

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.

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.

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

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?

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)

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)

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

Drug C

Clathrin “clumps”= non-functional

?

?

Adapted from Genes & Dev. 1998. 12: 3663-3674

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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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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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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

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

Drug C

Clathrin “clumps”= non-functional

BafilomycinBlocks endosome acidification?

?

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?

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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