Experimental Cell Biology 1: The basics
Cells are membrane-enclosed units of life. They Denatured proteins in the nucleus focussing on
have a metabolism, grow, divide and give the role of the nucleolus and its phase-separated
responses to environmental signals and can nature in protein quality control.
communicate internally and externally. The
genetic material in the cell is called DNA.
Metabolites are molecules that are produced or
altered by the cell.
The eukaryotic cell is larger than the
prokaryotic cell. The eukaryotic cell is multi-
cellular and has membrane-enclosed
compartments while the prokaryotic cell has no
membrane-enclosed compartments. Also, Upon heat stress, misfolded nuclear proteins
prokaryotic cells are biochemically flexible. entered the liquid-like GC phase of the nucleolus
where they were associated with certain proteins
The eukaryotic cell consists of: a nucleus, and adopted a state of low mobility. Storage in the
endoplasmatic reticulum, Golgi apparatus, GC phase prevented the irreversible aggregation
mitochondria, lysosomes, transport vesicles and of misfolded protein species, allowing their
chloroplasts. extraction and refolding upon recovery from
stress in an hsp70-dependent manner. 200
What is the difference between the nucleus different proteins were tested either entering the
membrane and the plasma membrane? nucleoplasm or from within the nucleolus.
The cell membrane is the biological membrane Disruption of the GC phase resulted in the
that separates the interior of all cells from the formation of stable aggregates of stress-
outside environment whereas the nuclear denatured proteins in the nucleoplasm which
membrane surrounds the nucleus, enclosing the exerted toxic effects. In conclusion, the liquid-like
genetic material. Besides the cell membrane is phase of the nucleolus functions as a non-
made up of one lipid bilayer structure and the membrane-bound protein quality control
nuclear membrane is made up of two lipid compartment. It is characterized by a chaperone-
bilayer structures. like capacity to temporarily store misfolded
proteins preventing their irreversible
The nucleus aggregation and maintaining them as competent
The nucleus is a specialized structure that is for hsp70-assisted refolding. Nucleoplasmic
separated from the rest of the cell by a double proteins exit the nucleolus upon refolding and
layer called the nuclear membrane. This nucleolar proteins resume their functional state.
membrane is continuous with the endoplasmatic
reticulum and has pores which permit the The endoplasmatic reticulum (ER)
entrance of large molecules. The nucleus The ER is a large, dynamic structure that serves
controls and regulates the activities of the cell many roles in the cell. These include protein
and carries the genes. synthesis, transport, protein folding, lipid and
steroid synthesis, carbohydrate metabolism and
The nucleolus calcium storage. The organelle itself looks like
The primary function of the nucleus consists of membrane-enclosed sacs also
ribosomal RNA (rRNA) transcription, processing known as cisternae in the
and ribosome unit assembly. The nucleolus is RER, and tubular structures
also involved in protein quality control. Cells in the SER. The RER is
have developed mechanisms that prevent the responsible for protein
formation of potentially toxic aggregates. During synthesis since
stress Hsp70 and other molecular chaperones it is covered with ribosomes.
assemble in the nucleus to protect the ribosomal SER is mostly found in liver
proteins against aggregation. A combination of cells since it’s involved in
fluorescence imaging, biochemical analyses and detoxification. The phospholipid membrane
proteomics was used to investigate the stress- encloses the cisternal space, which is continuous
,With the perinuclear space but separate from the
From the ER. The cargo proteins are packaged
cytosol.
into vesicles and secreted via exocytosis or for use
in the cell. The Golgi Apparatus is also involved in
The ER is also involved in mitochondrial fission.
lipid transport and lysosome formation.
Mitochondrial fission and fusion are coordinated
at the ER membrane contact sites (MCSs).
The mitochondria
Mitochondrial fission is initiatedby theER Mitochondria use aerobic
respiration to generate ATP,
which is used in the cell as a
source of energy. Mitochondria
are between 0.75 and 3 µm.
Mitochondria are involved in
signalling, cellular
differentiation, cell death and
maintaining control of the cell
cycle and cell growth. They are also involved in
Friedman et al. 2011 Science
fatty acid oxidation to generate energy.
Rough endoplasmatic reticulum The lysosome
The binding site of the ribosomes on the RER is A lysosome is a membrane-bound cell organelle
the translocon. A ribosome only binds to the RER that contains digestive/hydrolic enzymes. They
once a specific protein-nucleic acid complex break down excess or worn-out cell parts. They
forms in the cytosol, this happens when a free may also be used to destroy invading viruses and
ribosome begins translating the mRNA of a bacteria. If the cell is damaged beyond repair the
protein destined for the secretory pathway. The lysosome can help it to self-destruct, in a process
first amino acids encode a signal peptide that is that’s called apoptosis. The lysosome itself is very
recognized and bound by a signal recognition acidic and they carry around hydrolic enzymes
particle (SRP). Translation then pauses and the that need a low pH. They can break larger
ribosome complex binds to the RER translocon molecules into smaller molecules such as
where translation continues, forming a protein carbohydrates into sugars. This way the lysosome
into the RER lumen/membrane. The protein is can provide nutrients for the cell. There is also
processed in the ER lumen by an enzyme called evidence indicating that lysosomes are involved in
signal peptidase, which removes the signal processes such as secretion, plasma membrane
peptide. Ribosomes will then be released back repair, signalling and energy metabolism. But the
into the cytosol or stay associated with most essential role is the autophagic pathway. The
translocons. lysosomal membrane contains proteins that are
involved in the transport of substances into and
The Golgi Apparatus out of the lumen, acidification of the lysosomal
The Golgi is part of the endomembrane system in lumen, and fusion of the lysosome with other
the cytoplasm and packages proteins into cellular structures. Extracellular material that
membrane-bound vesicles before they are needs to be degraded enters the lysosome through
transported to their destination. It’s important the endocytic pathway while intracellular
for processing proteins, sorting, transport, and components are transported to the lysosome by
secretion and they contain a lot of glycosylation autophagy. Lysosomes can also secrete their
enzymes that attach sugar monomers to contents by fusing with the plasma membrane.
proteins as the proteins move through the This process is known as lysosomal exocytosis.
apparatus (modification). The Golgi also consists
of cisternae that are broken down into cis and Transport vesicles
trans compartments, which make up two A vesicle is a structure within or outside the cell
networks. The cis Golgi network is the first consisting of a liquid of cytoplasm enclosed by a
cisternal structure from which proteins are lipid bilayer. Vesicles are involved in the secretion
packaged into vesicles destined for lysosomes, and transport of materials within the plasma
secretory vesicles or the cell surface. The trans- membrane. Vesicles can also fuse with other
Golgi network acts as an early endosome in yeast organelles within the cell. Vesicles are crucial for
and plants. The Golgi itself is a collection and the distribution of material and information and
dispatch station of protein products received are closely related to the ER/Golgi.
,The cytoskeleton
The cytoskeleton is a system of filaments or
fibres that are present in the cytoplasm. The
cytoskeleton organizes components of the cell,
maintains the cell’s shape and is responsible for
the movement of the cell and the organelles
within. The three major types of filaments are
actin, microtubules and intermediate filaments.
Chloroplasts
Chloroplasts are structures within the cells of
plants and green algae that are the site of
photosynthesis to produce oxygen and ATP.
The cytosol
The cytosol is a liquid matrix found inside cells,
in eukaryotes, it’s enclosed with a cell
membrane. The cytosol consists of all the liquid
in the cell membrane except for the nucleus and
other organelles within the cell. All the liquid in
the cell + the liquid inside the organelles
together is called the cytoplasm. The cytosol
contains a broad range of molecules and many
chemical reactions take place. The cytosol is
very crowded and the high concentration of
molecules contributes to the rate of metabolism/
chemical reactions.
https://youtu.be/YM2X1c4K1x0
, Experimental Cell Biology: Energy metabolism
Cells need energy for metabolism for To produce energy from nutrients a few processes
biosynthesis, transport and movement. ‘The are involved.
energy for these processes is provided by the • Substrate-level phosphorylation (glycolysis)
breakdown of molecules in the diet such as • Citric acid cycle
carbohydrates, lipids and proteins. The energy • Oxidative phosphorylation
derived from the diet molecules is stored as
energy currencies. Common currencies of Glycolysis
energy in living cells are: Glycolysis is the metabolic pathway that converts
• Adenosine triphosphate (ATP) glucose into pyruvate. During this process, some
• Ion motive forces across membranes enzymes catalyse the reaction. First, there is the
energy required, 2 ATP gets used up whereas 4
ATP ATP + 2 pyruvates + 2 NADH comes out. The
When ATP is consumed in metabolic processes, it process happens in the cytosol and is very fast. It
converts to ADP or AMP, due to the loss of one or is also a very tricky process because ATP is
two phosphate groups. ATP is used for needed and this will not be possible when the cell
endergonic reactions, which means that the is deprived of energy.
reaction requires energy to proceed. What can
drive endergonic reactions, are: ion gradients
such as protons, sodium and potassium.
Researchers have studied how a eukaryotic model
can cope with the change of an environment with
high glucose levels and how the system fails which
results in metabolic malfunctioning and growth
arrest. The growth of yeast was measured
through flow cytometry and colony plating of a
Ion motive forces wild type and mutants that were unable to transit
The ion motive force is the sum of electrical and properly to excess glucose. The results showed
chemical potential differences (ion gradient) that the failure to cope with glucose was caused by
across the cell membrane and is the driving imbalanced reactions in glycolysis. In the failure
force of ATP synthesis (ATP synthase). When mode, the first steps of the glycolysis carry more
protons flow through a channel in the enzyme flux than the downstream steps which resulted in
the movement spins the protein (sort of like a constant low levels of ATP. In the mutants, only
wind turbine). This movement provides the 0,01% of the cells started growing and in the
energy to add a phosphate group to ADP to wildtype, 93% of the yeast cells started growing.
form ATP. Like most enzymes, but unlike most Models showed that the dynamics of phosphate is
ATP synthases, this enzyme can run a key determinant in a successful transition to
backwards within the cell. This ATP hydrolysis glucose and that the phosphate release through
can be the driving force to cause the enzyme to ATP hydrolysis reduces the probability of
release protons out of the mitochondria. The reaching an imbalanced state.
“ leak” back across the inner membrane is
needed to keep stimulating the membrane Pyruvate oxidation
potential to maintain the activity of the Pyruvate oxidation takes place in the
respiratory chain. mitochondrial matrix. During the pyruvate
oxidation, pyruvate gets converted into acetyl
CoA so it can enter the citric acid cycle. Pyruvate
is modified by the removal of a carboxyl Group
followed by oxidation and then attached to
Coenzyme A, which yields NADH. (Next page)
