This document provides a summary of the whole Experimental Cell Biology 2 course. **You can also find the separate lectures on my profile!
Topics that are discussed are:
Endothelial cells (leukocyte extravasation, chemokines, and stem cell homing), Basic techniques in cell biology (PCR, chroma...
The three Ras genes in humans (HRas, KRas, and NRas) encoding for small GTPases are among the most common oncogenes in human cancer; mutations that permanently activate Ras are found in 20 to 25% of all human tumors and up to 90% in certain types of cancer (e.g., pancreatic cancer). Ras inhibitors with good clinical efficacy have not been identified, despite decades of research. What would be another possible approach to prevent Ras from signaling?
Answer: A small molecule that activates the Ras GAP
2.
You wish to investigate which cellular factors are involved in the survival of Salmonella in human cells. RNA interference revealed that inactivation of various factors strongly decreased intracellular survival of Salmonella. To be sure that the factors you found with this approach are really involved in survival of Salmonella, you wish to carry out additional experiments. Which two techniques do you suggest to validate the findings from the RNA interference experiment?
Answer: qPCR and Western Blot
3.
What does the term \"kinome\" mean?
Answer: The complete set of kinases present in a certain cell/or organism.
4.
What does the abbreviation SAR mean related to characterization of drugs or drug targets?
Answer: Structure-activity relationship. The SAR provides information on how small changes in the structure of an inhibitor molecule affect the potency of the inhibitor.
5.
What is the Stokes’ shift in fluorescence?
Answer: The shift in wavelength between excitation and emission
6.
What is the best desciption of a quantum dot?
Answer: A Q-dot is an inorganic semiconductor fluorophore. Typical advantages are a narrow emission spectrum and bright emission.
7.
Some systems can become bistable. What is required?
Answer: Positive feedback
8.
Trehalose–phosphate synthase 1 has been revealed as an important enzyme in yeast. Why is this enzyme so important for growth of yeast?
Answer: This enzyme inhibits the upper part of glycolysis and stimulates the lower part of glycolysis. This contributes to a balanced glycolysis pathway, which is needed for growth.
9.
What is the apparatus for the detection and quantitation of peptides for proteomics analysis?
Answer: A high performance liquid chromatography system coupled to a tandem mass spectrometer (HPLC + MS/MS)
10.
A critical step in proteomics analysis is the digestion of proteins into peptides; trypsin is the most commonly used enzyme for this purpose. Trypsin cleaves which peptide bonds?
Answer: The carboxyl group of arginine, or the carboxyl group of lysine, and the amino group of the adjacent amino acid.
Content preview
Experimental Cell Biology II: Endothelial cells
Reasons for leukocyte transendothelial Transcellular and paracellular diapedesis
migration The last step of leukocyte migration is
• Immune surveillance crossing the endothelium with a process
• Inflammation called diapedesis. During diapedesis, the
• Cancer metastasis leukocyte crosses the endothelial barrier via
• Immune diseases (Rheumatoid Arthritis) either the paracellular or transcellular route.
• Stem Cell homing (after transplantation) During the paracellular diapedesis, the
• Atherosclerosis leukocyte crosses the endothelium through a
cell-cell junction, in-between two or three
Leukocyte extravasation adjacent endothelial cells. When migrating
This is the movement of leukocytes out of transcellular, leukocytes move through the
the circulation system towards the inflamed body of an endothelial cell, often close to the
tissue. This process of transendothelial junction where the luminal and apical
migration can be divided into several steps. membranes are nearby. This was investigated
1) Chemoattraction of leukocytes such as by using fluorescent proteins. Neutrophils
neutrophils and macrophages towards the and monocytes prefer the paracellular
infection site by cytokines/chemokines. pathway and T-cells prefer the transcellular
2) Neutrophils roll over the endothelial cells pathway.
and adhesion takes place
3) Crawling of the neutrophils and tighter
adhesion takes place
4) Diapedesis/endothelial transmigration
takes place.
It is possible to study this. Therefore, you
could isolate endothelial cells and veins
from the umbilical cord and study the
mechanism in vitro. With the help of certain
enzymes, the endothelial cells can be cut off
and collected in a petri dish and put in an The moment that the leukocyte has crossed
incubator to grow a monolayer of cells that the endothelium the endothelial pore is
represents the inner layer of the blood closed. The transmigration gap is closed
vessel. It is also possible to culture the using a contractile ring, enabling the
endothelial cells in a flow chamber. This way leukocyte to leave the endothelium and
the interaction between leukocytes and migrate into the tissue.
endothelial cells can be observed very
accurately. Chemokines
Chemokines attract cytokines. These are
Actin dynamics are essential for small glycoproteins (8-10kD) that are
transendothelial migration. With the help of immobilized on the surface of the
lattice light sheet microscopy, it can be endothelium. They act as a “road map” for
observed that the endothelium is activated cells and induce directional migration.
during inflammation. Adhesion molecules
are brought up and the actin filament
protrusions are coming out of the
monolayer to catch the leukocytes. The
molecule ICAM-1 goes around leukocytes
like a cup to catch them.
,The chemokine CXCL12 plays a role in migration in vitro. VE-cadherin blocking
attracting stem cells on the bone marrow increases stem cell homing after transplantation
endothelium during a stem cell transplant. in vivo.
Research has shown that when there is no
CXCL12 present on the endothelium, there
are no cells attracted compared to when
CXCL12 is present.
How can you promote stem cell homing?
There are two issues related to stem cell
transplants. The first one is that there are To determine whether leukocytes need to open
only small grafts available, which means that endothelial cell contacts during extravasation,
every stem cell counts. The second problem researchers generated mice with strongly
is that bone marrow reconstruction is very stabilized endothelial junctions and replaced VE-
slow and the patient is immunocompromised cadherin with VE-cadherin-α-catenin. These mice
due to this. were completely resistant to the induction of
vascular leaks, also neutrophil or lymphocyte
But how do immune cells cross the vascular recruitment into the inflamed lung and skin
barrier (5µm)? They do this by homing. tissue was strongly inhibited. These mice showed
Homing is the ability of stem cells to find the importance of the junctional route in vivo.
their destination or “niche”. The primary Surprisingly, lymphocyte homing into lymph
navigational clues used during homing seem nodes was not inhibited. Thus, the results showed
to be the same as those used in migration, that the junctional route (paracellular) is the
but homing may occur in any direction at any main pathway for extravasating leukocytes in
time. several, but not all tissues.
Research also showed that CD34+ cells prefer
transcellular migration since stem cells that
express CD34+ on their surface tend to
differentiate into T-cells in a thymic environment.
Therefore, the tendency for paracellular
migration is blocked.
But what triggers
Vascular endothelial (VE)-cadherin (CD144) this? Extravasation
VE-cadherin is an endothelial-specific Of leukocytes is
adhesion molecule located at the junctions Initiated by selectin-
between endothelial cells. VE-cadherin is of Dependent transient
vital importance for the maintenance and Rolling-type inter-
control of endothelial cell contacts. VE- Actions of the lumenal
cadherin glues endothelial cells together. In The surface of the endothelium. This facilitates
the bone marrow, when VE-cadherin is chemokine-dependent activation of leukocytes,
blocked, it makes the vascular wall which in turn triggers adhesion that is mediated
permeable so stem cells and other blood cells by the binding of leukocyte integrins to their
can pass through. endothelial ligands, such as ICAM-1. This results
in the diapedesis of the leukocyte through the
endothelial barriers. Long inflammatory
stimulation increases the levels of ICAM-1. T-cell
transmigration also depends on ICAM-1, but
seems to have different chemokine preferences,
though they do have the same integrin profile.
CX3CL1 Is present on the surface of the
endothelium and is recruited upon ICAM-1
Stem cell transplantation and irradiation clustering to trigger transmigration. ICAM-1
Vascular permeability is regulated by VE- clustering recruits the vesicle transport protein
cadherin, also after sublethal total body SNAP23, resulting in a local release of
irradiation (sTBI). VE-cadherin blocking chemokines and driving the migration of T-cells
promotes human CD34+ stem cell trans- in a transcellular manner.
,Vessel-on-a-chip technology
Dysfunction of the endothelial cells that line the
inside of all the blood vessels contributes to
many diseases, such as hypertension,
atherosclerosis, coronary artery disease, and
stroke. Consequently, considerable resources
have been devoted to research into the
molecular, cellular and physicochemical
determinants of blood vessel formation and
function. To address this, engineers managed to
form an environment where tissues and vessels
could be studied in 3D.
, Experimental Cell Biology II: Basic Techniques
Cell biology techniques: DNA manipulation and molecular cloning
• DNA/RNA techniques After the DNA is purified it can also be used to
• Protein techniques manipulate or clone the DNA.
• Knock-out To manipulate the DNA, the DNA can be split by
• Knock-down with RNAi enzymes to cut a specific sequence out of the
• CRISPR-Cas DNA. Restriction endonucleases are used to cut a
specific sequence out of the DNA at the specific
Cell tissues can be broken up by: restriction sites, these are about 4-8 bases long.
• Grinder or blender To cut the DNA, all restriction enzymes make
• Osmotic shock two incisions, once through each sugar-
• High pressure phosphate backbone of each strand and one of
• Detergent/enzyme treatment the DNA double helix. Once the piece is out of the
DNA, it can be used to insert inside another DNA
DNA techniques: sequence, which also has to be cut open. After
• Isolation putting two different sequences together in
• Manipulation/cloning solution, DNA ligase can be used to glue the ends
• Amplification of all the fragments together. After inserting the
• Investigation recombinant DNA into a host cell, the DNA can
be transcribed into mRNA and translated into a
DNA isolation protein by RNA polymerase.
To isolate DNA, a cell extract is prepared
during a process called lysis, where the To clone the recombinant DNA, the DNA requires
nucleus and the cell are broken open, thus a cloning vector. The DNA is inserted in the host
releasing the DNA. To free the DNA from its organism/cell. A polymerase chain reaction
proteins and other cell components, they are (PCR) is used to replicate the recombinant DNA
dissolved using enzymes such as Proteinase K sequence inside the host cell.
and detergents or an organic solvent. During
the precipitation step, the DNA is freed from Polymerase chain reaction
cellular debris. It involves using sodium ions to For the PCR, you need the DNA fragment,
neutralize the negative charge in DNA nucleotides, primers, buffer, and DNA
molecules (making them more stable). After polymerase. During the first step of the PCR, the
this, alcohol, ethanol or isopropanol is added to two strands of DNA are separated at a high
complete the precipitation and free the DNA. temperature, this is called nucleic acid
The DNA is then purified in a buffer solution or denaturation. In the second step, annealing, the
alcohol, which removes all the remaining temperature is lowered so that the primers can
cellular debris and unwanted material. bind to the DNA. After this, an intermediate
• There are kits available that often use a temperature is used so that the DNA polymerase
porous material for specific binding of DNA can make a new strand, this step is called
extension. These cycles are repeated 20-40
Agarose gel electrophoresis times.
This technique uses porous gel to separate
DNA fragments. The fragments are separated
by applying an electric field to move the
charged molecules through an agarose matrix,
negatively charged DNA moves to the positive
side of the electric field. The fragments are
separated according to size. Smaller DNA
fragments move further in the gel. The Reverse transcription PCR (RT-PCR)
separated DNA may be viewed with stain This is a modified version of PCR that amplifies
(DNA-binding dye, ethidium bromide), most RNA to investigate the mRNA in the cell. For
commonly under UV light, they can also be this, a reverse transcriptase is added to
extracted from the gel by cutting the wanted synthesize DNA from mRNA, after this a normal
DNA out. PCR reaction is performed.
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