Endocytosis: the Dynamic Nature of the Endomembrane System

Notes to Instructors are italicized on slides and in notes

2011 National Academies Northstar Institute for Undergraduate Education in Biology

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 structure Exocytosis

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

Diversity
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.
7/14/11

Class plan (single class session)

Before class: Students complete assigned reading 1. 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

Salmonella ?

Pathogens use the endocytic pathway to get into cells

e.g.: flu virus

Think-Pair-Share: Endocytosis
A) THINK INDIVIDUALLY (4 minutes) Read the terms on next slide Choose 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 lecture Modify your diagram as we discuss

Choose appropriate terms and create your own labeled diagram

Cargo Clathrin Coated pit Coated vesicle Endosome ER Golgi Lysosome Mitochondria Naked vesicle Plasma membrane Receptor

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, pinocytosis differences and similarities

Types of Endocytosis

Biology (2002) Campbell et al.

1. Multiple pathways of endocytosis 2. Molecular recognition between cell (receptor) and cargo (internalized object)

Receptor-mediated Endocytosis
Animated Video: Key Points 1. Recognition of SPECIFIC cargo 2. Multiple steps + compartments 3. Consequences for cargo
http://www.youtube.com/watch?v=lNKKg6hIBcM

Molecular Biology of the Cell (2002) Alberts et al.

Lecture continues
Cover steps clathrin acidification-2 steps Concepts: Molecular recognition Sorting cargo-receptor vesicle-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. Drug: Chlorpromazine (Drug C) Viruses: Ebola virus Vesicular stomatitis virus (VSV) Human Immunodeficiency Virus (HIV)

Adapted from: Bhattacharyya et al (2010)

Drug C: effect on normal cell endocytosis

C
nucleus

nucleus

Clathrin coated pits or vesicles Clathrin clumps

Images show clathrin localization inside cells.

Adapted from: Bhattacharyya et al (2010)

Drug C: effect on normal cell endocytosis

C
nucleus

nucleus

Clathrin coated pits or vesicles Clathrin clumps

Brainstorm in pairs (2 min): What steps of endocytosis might be affected by Drug C?

?
? Drug C

Clathrin clumps = non-functional

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

Drug C: effect on viral entry into cells

Drug C

% of cells infected

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.

Ebola

Adapted from: Bhattacharyya et al (2010)

Drug C: effect on viral entry into cells

Drug C

% of cells infected

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?

Ebola

A. Ebola only B. VSV only C. HIV only D. VSV and HIV

Drug C: effect on viral entry into cells

Drug C

% of cells infected

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 only B. VSV only C. HIV only D. Not enough information

Ebola

PREDICT THE OUTCOME

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.)

C V

Bafilomycin

?
? Drug C

Blocks endosome acidification

Clathrin clumps = non-functional

Bafilomycin: effect on viral entry into cells

Bafilomycin

Homework

This experiment is like the one in class, except cells were treated with Bafilomycin, which inhibits endosome acidification. Q1: What is the most likely method by which VSVg enters the cell? A. Clathrin-mediated endocytosis B. phagocytosis C. pinocytosis D. not enough information

% of cells infected

Q2: Justify your answer to Q1

Ebola

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?