Lecture 04, Sept 13/17 (Chris Loewen)

Cell Structure III

3rd edition: Ch 2 pg 36-48; Ch 3 pg 49-68
4th edition: Ch 2 pg 40-54; Ch3 pg 55-79

ER Junctions (cont'd):
-The first slide of the lecture summarizes the basic molecular structure of ER junctions
(mentioned during the last lecture; CELL II). You need to understand that there are 'bridging
complexes' that hold the membranes in close proximity and that the LTP's bind to receptors
on both the donor and acceptor membranes so that they can grab and release lipids as they
use their flexible 'hinge to swing between the donor and acceptor membranes. In this slide
'PS' was used as an example lipid only (i.e. all kinds of lipids get transferred between
membranes this way)

Mitochondria (up to 10um in length, 0.5-1um in width):

(Fig 2.28)
• Understand the classical structure (outer membrane, intermembrane space, inner
membrane/cristae, matrix.) and function of mitochondria (i.e. ATP production, apoptosis,
synthesize phospholipids).
• Understand that mitochondria are dynamic. They move on microtubules; undergo fusion and
fission/splitting
• The mitochondrial circular DNA (cDNA) contains only 37 genes, but the mitochondrion has
~1000 proteins; Therefore, most mitochondrial proteins are coded by nuclear genes and are
imported into the mitochondria.
• Mitochondrial import occurs at outer/inner membrane contact sites (eg. the components
involved are brought in close contact such that the transfer of the protein occurs over a short
distance between the membranes). Understand the 'stages' of mitochondrial import, using the
import of the ATP Synthase protein as an example:
I) Chaperones bind the polypeptide to be imported in the cytoplasm and transports it to the
mitochondrion.
II) A mitochondrial targeting sequence in the polypeptide directs it to the Translocase of the
Outer Membrane (=TOM) complex which is a large transmembrane channel that drives
the polypeptide through the outer membrane into the intermembrane space of the
mitochondrion.
III/IV) The small Translocon of the Inner Membrane (=TIM) complex, which is a peripheral
membrane, binds the polypeptide and delivers it to the large TIM complex, which is a
large transmembrane protein complex that inserts the polypeptide into the inner
mitochondrial membrane.
V) Polypeptide is released into the inner membrane and it folds into its native structure and
begins to function in generating ATP there.

Please note: do not need to know any of the other protein/gene names/numbers shown in
Slide 62 that make up the various complexes

• Understand that impaired mitochondrial structure and/or function can cause a number of
clinically important disorders. For example, impaired mitochondrial structure, which leads
 to impaired energy production, causes mitochondrial myopathy whose clinical signs (poor
muscle control/posture and cognitive impairment) are mostly the result of globally
inefficient ATP production in cells with high metabolic rates (eg. muscle cells and neurons);
Can be caused by mutations in the mitochondrial genes themselves or in nuclear genes
encoding for proteins imported into the mitochondria.

Peroxisomes (~1um):

• Small, membrane bound organelles; self-replicating; important in regulating metabolism in

all cells (eg. ß-oxidation-mediated breakdown of fatty acids which generates Acetyl-CoA
that is utilized in the citric acid cycle in the matrix of the mitochondria).
• All proteins are trafficked/transported to peroxisomes as they have no genetic material. This
is accomplished by:
1) A peroxisomal targeting signal sequence that targets cytosolic proteins to the
peroxisomal membrane by a peroxisomal targeting signal sequence in the protein;
2) Transfer of proteins from the ER by fusion with ER-derived vesicles with the
peroxisomal membrane.
• Peroxisomal defects affect many tissues, often severely (eg. Zellweger's Syndrome is caused
by an impairment of peroxisomal protein import. Deffective peroxisomes results in death
shortly after birth).

Cytoskeleton (Fig 2.30)

• Form a 3D network of protein filaments that act to maintain cell morphology, move
intracellular components (e.g. vesicles and organelles), and facilitate the movement/
migration of entire cells

Three major classes (initially classified by electron microscopy before proteins were
characterized and encoding genes were cloned):

I) Actin (~6nm diameter = thin filaments):

• 'Thinnest' cytoskeletal element; formed of filaments called fibrous actin (F-actin) which are
comprised of two chains of globular actin monomers (G-actin).
• Understand that filaments are dynamic with plus “+” (faster growing) ends and minus “-
“ (slower growing) ends. Capping proteins regulate the length and speed of growth.
• Filaments can be bundled together for different functions, and bundles are maintained by
different actin filament binding/stabilizing/cross-linking proteins (eg. α-actinin)
• Myosin motors can attach to F-actin to either initiate contraction (both in muscle and non-
muscle cells) or move cargo (eg. vesicles) along the bundled filaments.
• Focal adhesions are regions where the actin cytoskeleton binds/adheres to the extracellular
matrix (ECM) across the plasmamembrane (Fig 2.32).
• F-actin in 'stress fiber' bundles are linked to the cytoplasmic domains of
transmembrane integrins by a scaffold of stabilizing proteins (eg. vinculin and talin).
The extracellular domains of the integrins then bind extracellular matrix molecules (eg
fibronectin, laminin or collagen). These attachments are critical for cell adhesion and
migration (eg. during embryogenesis, tissue formation, wound-healing and many
organ system functions). This will be discussed in much more detail in the Cell
Adhesion and ECM lectures by Dr. Tanentzapf.
 • Analogous structures form in muscle cells that are disrupted in muscular dystrophy.
This will be discussed in more detail in the Muscle lectures by Dr. Moukhles
• Actin filaments can also be linked together into gel-like webs and networks by
'scaffolding' proteins such as cortactin and filamin in the outer portion/cortex of the cell.
These actin networks are critical for cell shape and, therefore, are very important for the
form and function of tissues.

II) Microtubules (25 nm in diameter):

• Hollow tubes formed by globular α- and β- tubulin heterodimers that have a plus “+”
growing end where dimers can be added to increase the length of the tubule
• Originate at the centrosome from γ-tubulin ring complexes = the minus “-“ end of the
tubule
o Centrioles are specialized microtubular structures within centrosomes, nucleate
microtubules and generate the long spindles that attach to chromosomes during mitosis
• Microtubules also critical for movement of organelles in the cell, and for the movement of
specialized cellular projections (eg. cilia and flagella) as well as chromosomal segregation
• 'Kinesin' motor proteins bind cargo and move along microtubules towards the plus “+” end
(usually to the outer aspect of the cell), whereas 'dynein' motor proteins move toward the
minus “-“ end of the microtubules (usually deep within cell/towards nucleus)
• There can be transfer of cargo from the microtubule system to actin filaments, particularly
near the plasma membrane (i.e. as occurs at terminal aspect of growing neurons = growth
cone which is what is shown in the left image on slide 70 where the microtubules are labeled
'Green' and the actin filaments are labeled 'Red')

III) Intermediate filaments (8-10nm in diameter):

• Filaments are 'intermediate' in diameter and formed by overlapping protein rods.
• Form structural scaffolds within the cytoplasm (eg. cytokeratins) and within the nucleus (eg.
lamins, located just under the nuclear membrane to give the nucleus its shape)

Please note: nuclear 'lamin' filaments are very different from extracellular 'laminin' fibers
located outside cells in the ECM (see 05 Lect, Adhesion).

Nucleus
• Understand nuclear structure: euchromatin (light patches in figure, unwound DNA that can
be efficiently transcribed); heterochromatin (electron dense, densely packed, not being
transcribed); nuclear envelope (ER network); nuclear lamina (lies underneath nuclear
envelope); nuclear pores, which are large protein assemblies embedded in the nuclear
envelope; and the nucleolus
o Clinical Note. Progeria – a genetic disorder caused by mutations in lamin proteins.
Although the mechanism is not entirely clear, these individuals age much more rapidly
than normal
o Nuclear pores have a size of 9-11nm and allow very small molecules to pass freely in and out
of the nucleus via the openings called the annulus. Larger molecules must be transported in
(import) and out (export) of the nucleus in a regulated fashion.
o Understand the process of nuclear import and export (a.k.a. 'The Ran GTPase Cycle'; slide 73,
modified from Fig 3.7 of the textbook) at the following level of detail:
• The major concept here is that the RanGTPase, is maintained in two different states
depending on location: in the cytoplasm it is GDP-bound ('off'); in the nucleus it is GTP-
bound ('on')
1) Capture: In the cytoplasm cargo proteins with a 'Nuclear Localization Signal' (NLS; often
three lysines = 'K-K-K' on the powerpoint diagram) interact with the importins
2) Import: leads to the movement of the cargo/importin complex through the nuclear pore
into the nucleus
3) Release (in nucleus): Once in the nucleus RanGTP binds the cargo/importin complex (via
importins' 'Nuclear Export Sequence [NES; often a string of leucines]) and the cargo is
released
4) Export: RanGTP/Importin complex are then exported out of the nucleus
5) Release (in cytoplasm): In the cytoplasm the GTPase activating proteins ('RanGAPs')
convert RanGTP to RanGDP. This turns the Ran 'off' such that it releases the importin
proteins so that they can begin the cycle again with new cargo proteins
6) Diffusion: RanGDP, which is small in size diffuses back through the nuclear pore
7) Ran activatin: In the nucleus, Ran Guanine Exchange Factors (RanGEFs) convert
RanGDP to RanGTP by facilitating the exchange of GDP for GTP so that it is once more
'on' and ready to export importins (or any other proteins in the nucleus with an NES
sequence)

Please note: The positions of RanGAP and RanGEF are critical for cycle function; they
cannot diffuse freely through the nuclear pores because they are too large

Ribosome biogenesis (Fig. 3.11)

• Understand that nuclear import is required to bring individual ribosomal proteins into the
nucleus after binding to importins; Ribosomal subunit assembly then occurs in the nucleolus;
subsequently, ribosomal subunits, whose proteins contain NES signals, are exported from the
nucleus in a RanGTP-dependent process (slide 74; Fig 3.11)

Cell Cycle
o Know the major events of the cell cycle (Fig 3.12) including although not discussed in class,
the stages of mitosis (see Fig 3.16).
o Understand that there are checkpoints in the cell cycle that cells move through when the
appropriate combination of cyclins (Cln) and cyclin-dependent kinases (CDKs) are present
and thus provide a positive signal of the cell's readiness to move to the next stage in the cell
cycle.
o Know the one specific example provided in the lecture where DNA damage (eg. genetic
mutation) activates the transcription factor 'p53' which then induces production of the p21
protein that then provides an inhibitory signal that inhibits the CyclinE/CDK2 complex such
that the cell does not move from G1 to S (i.e. it is a cell cycle 'Checkpoint'). This gives the
cell a chance to repair the DNA damage/mutation. If it cannot repair the problem, p53 can
then direct the cell to undergo apoptosis via the 'intrinsic' apoptotic pathway. This often
 occurs in proliferating epithelial cells within the epidermis of the skin that have been
exposed to DNA damaging UV rays from the sun.
o Given its critical function p53 is often called the 'Guardian of the Genome'. If p53 is mutated
cell cycle checkpoints and mutation-induced apoptosis is impaired. This facilitates the
survival of mutated cells and can contribute to tumor formation. Thus, p53 is known as a
'tumor suppressor' and it is mutated in many cancers.

Apoptosis
• Active 'programmed cell death'
• Reduces inflammation as there is much less cellular debris released than occurs after
unprogrammed, passive death (i.e. 'necrosis').
• Important in controlling cell numbers in development (eg. cell removal during toe and finger
formation in the hands and feet so we don’t have webbed digits) and in responding to
cellular damage or pathological processes by clearing cells from damaged and/or injured
tissue.
• Two major types of Apoptosis lead to internal macromolecular breakdown by a group of
common proteolytic 'executioner caspases' enzymes:
-Extrinsic pathway - initiated by extracellular death ligands (ie. come from outside the
cell and therefore are 'extrinsic'; often they are released by immune cells) that bind to
death receptors and initiate signaling pathways that activate the executioner caspases
that cause a breakdown of the cytoskeleton, membrane blebbing and chromosomal
digestion cleavage and cell death.
-Intrinsic pathway - initiated by intracellular stresses such as DNA damage and ER
stress (therefore everything occurs inside the cell = 'intrinsic') that initiate the release
of cytochrome c from the mitochondria which activates the 'apoptosome' (APAF1) that
ultimately activates the same executioner caspases in the intrinsic pathway.
- There is some cross talk between the two pathways such that the activation of one pathway can
ultimately activate the other pathway. This increases the robustness of the response.

Please note: the Apoptosis Movie is posted under “Useful Links”