Biology 2B03 Midterm 2 Notes
Module 5 - Cell Adhesion
Cell Adhesion - Stable Interactions
Cell adhesion plays a role in allowing cells to be held together, and was a vital
process for the evolution of multicellular organisms. Specialized cells group together
to perform a specific task.
During embryogenesis we see cells recognizing each other through the use of cell
adhesion molecules (CAMs) which allow cells to segregate into distinct tissues.
CAMs are subdivided into classes based on their functions and interactions. After
aggregation these cells form specialized cell junctions. These stabilize cell-cell
interactions and promote communication between adjacent cells.
CAMs consist of cadherins, Immunoglobulin (Ig) family, integrins and selectins.
Single-celled layers of cells in the single plane, cells in contact with neighbouring
cells through cadherins, which allow specificity of adhesion, like cells recognizing
other cells. Within epithelial cells, the matrix at bottom that is at cellular structure
there are also transmembrane proteins that interact with the basal lamina.
Apical surface vs Basal surface
The apical surface contain microvilli that allow communication and adhesion.
All permanent interactions that are not released and rebuilt.
Adhesive Interactions and Motile Cells
Endothelial cells (specialized epithelial cells) form the inner lining of the blood
vessels. At some point the leukocytes must respond to an infection and move out of
the circulatory system to damaged or infected tissue and break through the
interactions that are holding the epithelial walls together. The movement out of the
blood vessel into damaged or infected tissue is known as extravasation.
Inflammatory response: Local infections or injury rapidly attract white blood cells to
the affected region. Leukocyte are broken up into three major categories;
granulocytes which contain toxins for killing pathogens. Second is monocytes which
are responsible for phagocytosis. Finally there is lymphocytes which are responsible for the lysis of virally infected cells and tumour cells as well as a role in the immune
Extravasation of Neutrophils
The infection has to cause a signal in order to enact a response, however we will
focus mainly on the response.Extravasation is a 5 step process. The process
involves capture of a leukocyte, causing the leukocyte to slow down and move
slowly. As the leukocyte continues to roll it eventually stop and form a firm adhesion.
In the final step, transmigration, the leukocyte splits apart two endothelial cells and
moves through the endothelium barrier.
Whenever we experience a cut there is a swelling and redness associated at this
site. This is due to the accumulation of blood cells, fluids and leukocytes flowing
through the hole in the endothelium barrier that allows the leukocyte to flow through.
Cytokines released by macrophages causes P-selectin to be released from vesicles
within the cell onto the cell membrane of the endothelial cell. TNFα is an example of
such a cytokine and is been shown to stimulate endothelial cells to reveal the
surface adhesion molecules P-selectin, and E-selectin. The selectin ligand is a specific carbohydrate that is always expressed on the neutrophil. Note that the
endothelium always has selectins, but the number of selectins increases after a
infection is present.
Interactions between these selectins and neutrophil cause the cell to move more
slowly. Role of ICAMs?
After slowing down we have to achieve full adhesion (full stop) before we can
undergo transmigration. The activation of cell molecules to create adhesion is done
through cytokines (IL-8/PAF) bound to membranes of endothelial cells to interact
with the cytokine receptors expressed on the neutrophils. The cytokines are
expressed pretty much all the time (they sometimes increase in expression) but they
cannot interact with the neutrophil unless it is slowed down through the slow rolling
process. Once the interaction between the cytokine and receptor occurs integrin
proteins on the neutrophil are moved into the membrane.
There are a specific set of interactions from selectin proteins located on the
endothelial cells that introduce a transcriptional change within the leukocyte which
allow it to produce changes later on.
The integrin protein changes its conformation in response to the presence of the
cytokine (how?) which allows it to become unfolded and interact with the ICAMs
present on the endothelial cells. ICAMs located on the endothelial cells are found to
interact with integrins after selectins slow down the movement of neutrophils.
After the slowing movement has occurred it allows for other interactions to occur.
Special cell adhesion molecules are expressed only at the site of infection on
endothelial cells called ICAMs. These create stable interactions with the slowed
leukocytes. Once the cell has stopped it allows the activation the signaling pathways
on the neutrophil.
Firm adhesion breaks, allowing the cell to move through the gap. This can only
happen after the leukocyte has stopped moving. We know very little about the lateral
movement of endothelial cells and the repair of the hole afterwards.
Module 6 - Cell Division
The cell cycle is an ordered series of events that produces two identical daughter
cells from a single cell. There are two main events. Chromosome replication/duplication wherein DNA synthesis occurs and cell division where the
nuclear envelope breaks down into vesicles which are segregated into daughter
cells, chromosome segregation occurs at an even distribution, and cytokinesis where
the organelles are sorted into daughter cells (this can be done asymmetrically)
occurs. The cell division is tightly regulated for the normal development of
multicellular organisms. Loss of the cell cycle control leads to cancer.
The picture to the right is of shows 7 different stages of the cell cycle. In the picture
green is microtubules, and blue is DNA. 1. G2 interphase 2. prophase, 3.
prometaphase 4. metaphase 5. anaphase 6. telophase 7. Cytokinesis
The chromosomes have not condensed, DNA replication is occurring, and the
nuclear envelope still exists.
During prophase I we can see the chromosomes begin to condense and the nuclear
envelope is beginning to dissolve. The centrosome have divided into two
centrosomes, but are located close to one another.
We see the two spindles have formed and are moved to the opposite poles. The
spindle apparatus is beginning to assemble. The chromosomes are more or less
completely compacted and are in a disorganized state within the centre of the cell.
The nuclear envelope is gone. Prometaphase is very short. Chromosome
attachment to spindle occurs.
We can see the microtubules condensed into two poles, and the condensed
chromosomes are lined up along the equator, the nuclear envelope is missing. Once
every chromosome has been linked with a microtubule in the spindle apparatus for
each pole, that is the cue for anaphase to begin.
The condensed chromosomes are being pulled apart into the daughter cells.
Microtubules are moving apart to the separate poles of the cell.
The chromosomes have migrated into the centre of the new daughter cells. The
nuclear envelope is also reforming. In the middle we can see the microtubules are
changing their arrangement to facilitate the pushing apart of the cells. In the actin filaments in the membrane of the cell constrictions are occurring causing the
beginning of cytokinesis. Spindle disassembly occurs.
Cell cycle phases
Regular mitotic division is the alternation between chromosome
replication/duplication and cell division. There are 5 cell cycle phases are growth
(G1), synthesis (S), growth (G2), M (Mitosis). There is an additional phase known as
G0 where the cell has exited the cell cycle. This can be temporarily for days/weeks
(most cells) or permanently (nerve/muscle cells).
Cell Cycle Control
The cell cycle is controlled through regulated phosphorylation of heterodimeric
protein kinases made up of 1. cyclins (regulatory subunit) and 2. cyclin-dependent
kinases (catalytic subunit) together forming Cyclin-Cdk complexes.
Cyclins also undergo regulated degradation: ubiquitination of cyclins and proteolysis
via E3 ligase complexes such as APC, SCF occurs, so cyclins can be removed from
the cell through degradation.
We can break down the cyclin-CDK complexes into 3 types. G1 cyclin-CDK, S-
phase cyclin-CDK and mitotic cyclin-CDK which are responsible for causing the cell
to enter into each of the three respective phases; G1, S and mitosis.
G1 Cyclin-CDK complexes
Prepares the cells for S-phase. Induce synthesis of proteins required for DNA
synthesis (includes proteins such as DNA polymerase, DNA binding proteins such
as histones etc). Induces degradation of S-phase inhibitors, thus activating S-Phase
cyclin-Cdk complex to stimulate cell entry into S-phase.
Targets of Phosphorylation
- S-phase inhibitors (they must be phosphorylated for degradation by
- Transcription factors required for synthesis of S-phase proteins
S cyclin-CDK complexes
Activates pre-replication complex. Assembly of pre-replication complex to activate
initiation of DNA replication. Prevents reassembly of new pre-replication complexes.
This last step is particularly important in order to prevent a second round of DNA
synthesis, so chromosomes are only replicated once. M cyclin-CDK complexes
Many proteins are phosphorylated in order to allow each of the functions mentioned
above in the stages of mitosis (breakdown of nuclear envelope, chromosome
condensation, assembly of mitotic spindle apparatus, alignment of chromosomes at
metaphase plate, activation of anaphase promoting complex (APC) at different steps
etc) and some proteins are degraded. (cyclins, anaphase inhibitors etc)
Identification of cyclin-CDK
These experiments identified a maturation promoting factor that could induce cell
division. At different stages of the cell cycle, different proteins have higher
concentration. The concentrations of proteins were monitored at these stages of the
cell cycle and then correlated to various physiological changes observed in the cell.
This is how the maturation promoting factor or Mitosis Promoting Factor (MPF) was
first identified. It is now known that MPF = Cyclin and Cdk heterodimer (specifically
mitotic Cyclin B and Cdk).
Identification of Cell Cycle Genes
Genetic technique were used to identify the genes that code for cell cycle regulators
in many models, including budding yeast and fission yeast. In particular study of
temperature sensitive cell-cycle mutations have been performed. To identify these
genes for temperature control, organisms are mutagenized using radiation or
chemical mutagens which create mutations are random; anything can be mutated.
Each mutation can potentially have different phenotype. A genetic screen looks
through all of these phenotypes for those of particular interest.
Temperature Sensitive Mutants
A wild type gene that codes for a protein and functions at normal temperatures
(25°C) and elevated temperatures (35°C). In contrast a temperature-sensitive
mutation in a gene codes for a protein that folds and functions at the normal
permissive temperature, but does not function at the elevated, restrictive
temperature. Yeasts are haploid organisms, only one copy of each gene is present.
If a gene is essential for viability, growth or replication, a loss of function mutant will
lead to death. As a result, the mutation could not be isolated. In contrast, an
organism with a TA mutant can grow and develop at permissive temperatures, but
the effects of loss of function can be studied at the elevated temperature.
There are two major phenotypes of TS mutants
Recessive (absence of wild-type function)
Dominant (increased wild-type function)
Note on nomenclature
S. pombe S. cerevisiae
Wild type gene cdc2+ CDC28 Recessive mutant gene cdc2- cdc28
Gene product Cdc2 Cdc28
How do we Isolate TS Mutants?
First a large collection of the organism is made to undergo mutation with chemicals
or radiation., creating a population of yeast cells with mutations in many different
places. These yeast are then grown at the permissive temperature. After a while the
temperature is shifted to the restrictive temperature, and researchers begin to
identify those that cannot divide or divide too quickly. These defective cells are Cell
division cycle mutants or CDC for short.
CDC2 Mutants in S. Pombe
An example of such an irregular phenotype for mutated cells was demonstrated in
the mutation causing loss of function of cdc2. This resulted in incredibly elongated or
small (wee) cell sizes. Long cells were indicated by cdc2 mutants while wee cells
were indicated by cdc2 cells. The wild type/regular phenotype was cdc2 . The failure
in this system is associated with failure to undergo mitosis (long cell) or premature
mitosis (wee cell). Further research showed that this cdc2 protein forms a
heterodimer with cdc13. Cdc13 is also required for mitosis entry, and the Cdc2-
Cdc13 heterodimer = MPF or mitotic cyclin-Cdk. They also function as a G and S
phase cyclin-Cdk. Cdc13 is homologous to a cyclin, and cdc2 would then function as
A deficit of cdc25 was found to create elongated cells (increased G2) while an
excess of cdc25 was found to create small cells (decreased G2). Cdc25 was found
to stimulate mitosis by activating MPF.
An excess of Wee1 was f