Experimental
cell biology 2
Minor: Biomolecular
science track neuroscience
Claire Snel
,Inhoudsopgave
Lecture 1 – Endothelial cells ............................................................................................................................. 2
Lecture 2 – Basic techniques ............................................................................................................................ 8
Lecture 3 – Proteomics ................................................................................................................................... 18
Lecture 4 – Host factors .................................................................................................................................. 30
Part 1 Introduction: why do we need new antibiotics ...................................................................................... 30
Part 2 Identification of host target genes to limit salmonella replication ........................................................ 33
Part 3 Short primer: small GTPases .................................................................................................................. 39
Part 4 Dissecting the molecular mechanism .................................................................................................... 43
Lecture 5 – Human microbiome ..................................................................................................................... 52
Part 1 Intro and biogeography ......................................................................................................................... 52
Part 2 Health and disease ................................................................................................................................. 62
Part 3 Modulation of the human microbiome .................................................................................................. 69
Lecture 6 – Single-cell gene expression .......................................................................................................... 76
Part 1 Why is it important to study gene expression in single cells? ................................................................ 76
Part 2 Why do we study gene expression in living cells? .................................................................................. 88
Lecture 7 – Fluorescence microscopy ............................................................................................................. 96
Part 1 Fluorescence and fluorescence microscopy ........................................................................................... 96
Part 2 Intraflagellar transport and microtubule dynamics in C. elegans chemosensory cilia ........................ 103
Lecture 8 – Apoptosis ................................................................................................................................... 109
Lecture 9 – Glycolysis ................................................................................................................................... 115
1
,Lecture 1 – Endothelial cells
Introduction
- Sanquin internships – good option for your internship > wright a personal email
- First 9 slides > story about lecturer itself, what he did
- He continued research on leukocyte trans endothelial migration
How do cells know where to go and how do they do that?
- Chemokines
➢ Cells, like neutrophils (in green) can go upstream the
bloodstream and attach to endothelial wall
➢ When vessels are damaged and a local
inflammation happens > the blood vessels
become activated by the alarm signals
(partly released by bacteria themselves:
antigens)
➢ As a result, leukocytes are recruited through the vessel wall and migrate towards the
infection to clear it
➢ Then they migrate back through the lymphatic vessels to be destroyed and the
wound can heal
➢ The question is: how do they manage to get out of the blood vessel while leaving the
blood vessel intact and not causing leakage from the blood vessel?
Leukocytes leave the blood vessel and crawl towards the inflammation
= Leukocyte trans endothelial migration
o Immune surveillance
o Inflammation
o Cancer metastasis
o Immune diseases (rheumatoid arthritis)
o Stem cell homing (after transplantation)
o Atherosclerosis
- Capillaries is the place where the migration takes place > vesicle interaction > issues
with capillaries is important
2
, How do immune cells breach the vessel wall
➢ Transendothelial migration
➢ Skin of bacteria activates the endothelial cells of
the tissue
➢ The neutrophils bind to endothelial cells very
strong
➢ They start rolling and crawling
➢ They go in between the endothelial cells or go
through the endothelial cells
Endothelial cells harvested from umbilical cord
- Umbilical cord: 2 arteries and 1 vein
- Human umbilical vein endothelial cell >
HUVEC > vein can be used to get
endothelial cells out > add enzyme that
takes vein out
No inflammation > nothing happens
Stimulate endothelial cells (by bacteria) >
leukoctyes attach
Out of bloodstream trough tissue >
outervagation
1. Rolling
2. Adhesion
3. Transmigration
a. Paracellular
b. Transcellular
>> how does the actin look
Expression of lifeACt-GFP into endothelial cells > cytoskeleton of endothelial cells is visible
Leukocyte transendothelial migration is involved in:
- To protect the human body from invading pathogens: immune surveillance and local
inflammation. Leukocytes attack the pathogens
- Homing of stem cells after stem cell transplantation
o Since chemotherapy is harmful for fast dividing cells, not only cancer cells will
be killed but also stem cells
o To counteract this problem, stem cells are first taken out of the patient and
stored in liquid nitrogen (cold temperatures)
o After the chemotherapy the stem cells are transfused back into your vein
o The stem cells have the capacity to find their way back to where they should
be, for example in the bone marrow
o In the last part of finding their way back, they cross the vessel wall
3
cell biology 2
Minor: Biomolecular
science track neuroscience
Claire Snel
,Inhoudsopgave
Lecture 1 – Endothelial cells ............................................................................................................................. 2
Lecture 2 – Basic techniques ............................................................................................................................ 8
Lecture 3 – Proteomics ................................................................................................................................... 18
Lecture 4 – Host factors .................................................................................................................................. 30
Part 1 Introduction: why do we need new antibiotics ...................................................................................... 30
Part 2 Identification of host target genes to limit salmonella replication ........................................................ 33
Part 3 Short primer: small GTPases .................................................................................................................. 39
Part 4 Dissecting the molecular mechanism .................................................................................................... 43
Lecture 5 – Human microbiome ..................................................................................................................... 52
Part 1 Intro and biogeography ......................................................................................................................... 52
Part 2 Health and disease ................................................................................................................................. 62
Part 3 Modulation of the human microbiome .................................................................................................. 69
Lecture 6 – Single-cell gene expression .......................................................................................................... 76
Part 1 Why is it important to study gene expression in single cells? ................................................................ 76
Part 2 Why do we study gene expression in living cells? .................................................................................. 88
Lecture 7 – Fluorescence microscopy ............................................................................................................. 96
Part 1 Fluorescence and fluorescence microscopy ........................................................................................... 96
Part 2 Intraflagellar transport and microtubule dynamics in C. elegans chemosensory cilia ........................ 103
Lecture 8 – Apoptosis ................................................................................................................................... 109
Lecture 9 – Glycolysis ................................................................................................................................... 115
1
,Lecture 1 – Endothelial cells
Introduction
- Sanquin internships – good option for your internship > wright a personal email
- First 9 slides > story about lecturer itself, what he did
- He continued research on leukocyte trans endothelial migration
How do cells know where to go and how do they do that?
- Chemokines
➢ Cells, like neutrophils (in green) can go upstream the
bloodstream and attach to endothelial wall
➢ When vessels are damaged and a local
inflammation happens > the blood vessels
become activated by the alarm signals
(partly released by bacteria themselves:
antigens)
➢ As a result, leukocytes are recruited through the vessel wall and migrate towards the
infection to clear it
➢ Then they migrate back through the lymphatic vessels to be destroyed and the
wound can heal
➢ The question is: how do they manage to get out of the blood vessel while leaving the
blood vessel intact and not causing leakage from the blood vessel?
Leukocytes leave the blood vessel and crawl towards the inflammation
= Leukocyte trans endothelial migration
o Immune surveillance
o Inflammation
o Cancer metastasis
o Immune diseases (rheumatoid arthritis)
o Stem cell homing (after transplantation)
o Atherosclerosis
- Capillaries is the place where the migration takes place > vesicle interaction > issues
with capillaries is important
2
, How do immune cells breach the vessel wall
➢ Transendothelial migration
➢ Skin of bacteria activates the endothelial cells of
the tissue
➢ The neutrophils bind to endothelial cells very
strong
➢ They start rolling and crawling
➢ They go in between the endothelial cells or go
through the endothelial cells
Endothelial cells harvested from umbilical cord
- Umbilical cord: 2 arteries and 1 vein
- Human umbilical vein endothelial cell >
HUVEC > vein can be used to get
endothelial cells out > add enzyme that
takes vein out
No inflammation > nothing happens
Stimulate endothelial cells (by bacteria) >
leukoctyes attach
Out of bloodstream trough tissue >
outervagation
1. Rolling
2. Adhesion
3. Transmigration
a. Paracellular
b. Transcellular
>> how does the actin look
Expression of lifeACt-GFP into endothelial cells > cytoskeleton of endothelial cells is visible
Leukocyte transendothelial migration is involved in:
- To protect the human body from invading pathogens: immune surveillance and local
inflammation. Leukocytes attack the pathogens
- Homing of stem cells after stem cell transplantation
o Since chemotherapy is harmful for fast dividing cells, not only cancer cells will
be killed but also stem cells
o To counteract this problem, stem cells are first taken out of the patient and
stored in liquid nitrogen (cold temperatures)
o After the chemotherapy the stem cells are transfused back into your vein
o The stem cells have the capacity to find their way back to where they should
be, for example in the bone marrow
o In the last part of finding their way back, they cross the vessel wall
3
