Transcript Endocytosis (PowerPoint) Midwest 2011

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
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•
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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
% of cells infected
Drug C
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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
% of cells infected
Drug C
•
•
•
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•
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
% of cells infected
Drug C
Ebola
•
•
•
•
•
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
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
D
V
B
C
A
C
V
Bafilomycin
?
?
Drug C
Clathrin “clumps”
= non-functional
Blocks endosome
acidification
Homework
Bafilomycin: effect on viral entry into cells
Bafilomycin
This experiment is like the one in class, except
cells were treated with Bafilomycin, which
inhibits endosome acidification.
% of cells infected
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
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?