Fig I
In this electron micrograph identify all the different unit membranes seen and the
compartments they enclose/make up.
a. Inner mitochondrial membrane ( encloses matrix or intra-membrane space)
b. outer mitochondrial membrane (with inner, enclose the inter-membrane space),
c. plasma membrane – each encloses a cell,
d. nuclear envelope – inner layer – encloses the nucleus
e. outer layer – (with inner) enclose a lumen continuous with that of the ER,
f. ER membranes, enclose cisternae
g. unit membranes surrounding peroxisomes.
Which of these different unit membranes are continuous with each other?
Nuclear envelope, ER membranes and possibly plasma membrane
For the insert, identify the different compartments seen. Can you resolve the trilaminar
appearance/structure of the unit membrane – what do these “3” layers correspond to
biochemically?
The different compartments seen are the cytoplasm on each side and the extraq-cellular
space in between the two membranes.
Each membrane can be resolved into dark:- phosphate head on inside of cell, light – fatty
acid tails- tail to tail, dark phosphate head on outside
Fig II
This is a high power light micrograph of a cell in tissue culture ( i.e. grown in a petri-dish
or in vitro).
Note the dark staining (hetero-) chromatin in the nucleus and the Cytocentre- a pale
staining area close to the nucleus.
The most important oprganelles situated here would be the Golgi apparatus and the
centrioles.
Which organelle/s are situated in this peri-nuclear cytocentre?
After answering, compare with the electron micrograph in Fig III ( which is also of the
cytocentre).
Fig III
This is an electron micrograph of the cyto-centre, enclosed within the dotted lines. It is
tan area of the cytoplasm next to the nucleus.
Identify the different organelles and structures (some are parts or products of organelles)
labelled.
Golgi apparatus, lysosomes, SER, ribosomes, microtubules, centriole, centriole spindle
Specifically what are X and Y? What are they part of?
X and Y are the trans and the cis faces respectively of the Golgi apparatus.
What is Ce? And what is CeS?
Ce is a centriole
CeS is a centrioles spindle
Also identify the other labelled structures outside the cytocentre.
N = nucleus, M = Mitochondria, ER = rough endoplasmic reticulum
Fig IV
This is a scanning electron micrograph ( reflection, not transmission), following freeze
fracture of a cell which shows the outer surface of the nuclear envelope.
Note the Nuclear pores.
What makes up the nuclear pores?
Groups of proteins forming channels – in this particular conformation – an outer ring of 8
proteins, radial spokes and a central plug.
What function do the nuclear pores serve – identify one type of molecule which passes
outward through a nuclear pore? Identify one type of molecule which passes inwards
through a nuclear pore?
The nuclear pores act as channels to allow passage of molecules in and out through the
pores.
An example of a molecule which passes outward is a m-RNA molecule.
An example of a molecule which passes inwards in a DNA-binding protein like a
transcription factor. (others found on Prof Cuschieri’s notes on the web).
Fig V
This is a transmission electron micrograph of a section through a nuclear pore.
Note the continuity of the External leaf of the Nuclear Envelope with the rough
endoplasmic reticulum. Note the continuity of the inner and outer leaves of the nuclear
envelope at the nuclear pore.
With which other organnellar space is the “lumen” of the nuclear envelope continuous
with? Could it therefore contain glycosylated proteins (glycoproteins) or not?
It is continuous with the lumen of the cisternae of the RER – therefore it could easily
contain glycosylated proteins.
Fig VI
Scanning electron micrograph of the nucleus and RER
Identify the structures labelled and answer the questions.
Structures labelled are : Cr – Chromatin
I - Inner layer of nuclear envelope
E – External layer of nuclear envelope
R – Rough Endoplasmic reticulum
The round structures on the surface of E are Ribosomes. The round structures on the
surface of R are ribosomes
The arrows on I are pointing to are nuclear pores.
FigVII
High magnification transmission electron micrograph of part of a nucleus.
Note the chromatin including Heterochromatin (labelled – CH) and the fine granular
(unlabelled) euchromatin in the rest of the nucleus.
a. What does the chromatin consist of ?
Chromatin consists of Chromosomal DNA and associated proteins.
b. How does the appearance of chromatin correlate with the activity of DNA?
Euchromatin ( which is less electron dense and less condensed) normally includes
active DNA ( ie DNA which is actively being transcribed) whilst heterochromatin (
more condensed and electron dense) is usually largely inactive.
The nucleolus is shown consisting of granular (GZ) and fibrillar(FZ) regions
What are the differences in composition and functions of the two zones?
The granular zone is made up largely on ribosomes as they are being formed from
ribosomal RNA and proteins, whilst the fibrillar zone is largely made up of ribosomal
RNA genes transcribing ribosomal RNA.
What is the name for the parts of chromosomes found in the nucleolus. How many
human chromosome pairs contribute to this structure?
The short arm satellites of acrocentric chromosomes are the nucleolar organising region
of the nucleolus. 5 human pairs of chromosomes contribute to the nucleolus.
Fig VIII
A low power transmission electron micrograph of a cell in mitosis.
Which stage of mitosis is the cell at?
Metaphase
How many centrioles are there in the cell at this stage?
4
To what do the mitotic spindle fibres lead at each of their ends?
At one end they attach to the centromere of the chromosomes and at the other end to the
centrioles.
What are the spindle fibres made of? Microtubules which are polymers of tubulin.
Fig IX
This is an electron micrograph showing part of a cell in mitosis
What of the list of structures can be identified in this micrograph.
The structures identified in the micrograph include :- microtubules, centrioles and the
rough endoplasmic reticulum. The other structures cannot be seen.
For the non-visible structures: Nuclear envelope – this has been dissolved/lost during mitosis.
Kinetochores - are the basal bodies of cilia or flagellae and are not visible in this section.
They are not to be confused with centrioles.
Actin filaments – are generally too small to be seen unless bunched together as in high
power views of muscle cells.
Nucleolus – has also been dissolved as a visible structure during the process of mitosis.
A = chromosomes
B = centriole
Fig X
Observe the positions of the chromosomes and the microtubules and centrosomes in the
mitotic cell.
No answers necessary – all were given
Before looking at the notes, can you identify the different phases?
Fig XI
High power electron micrograph showing a high power detail of a centriole.
What are the arrows pointing at?
The arrows are pointing at groups of microtubules – which are forming the spindle
Which part of the cell cycle would you suspect this is in?
This would likely be metaphase or anaphase
What special function does the centriole play in ciliated cells?
In ciliated cells, the centriole acts as an organiser for the cilia basal bodies, which
themselves then produce the cilia.
Fig XII
This is a low power electron micrograph of a ciliated epithelium from the trachea.
a. Note that the central microtubule pair in the cilia are all oriented in the same
direction. What is the significance of this observation?
This results in all the cilia sweeping in the same direction, since it is the central
microtubule pair which controls the direction of bending ( or beating) of the cilium.
b. If a section were taken deeper to show the basal bodies of the cilia, how many
central and peripheral microtubules would be visible?
A section taken through the level of the basal bodies would show 9 peripheral triplets
and no central pair.
c. Where else in the body is a ciliated epithelium found – what is its function?
A ciliated epithelium is also found in the fallopian tubes or oviducts – and its function
is to guide the ovum towards the uterus.
Fig XIII
High power view of a transverse section though a ciliated epithelium.
Identify the structures labelled A to G in the associated diagram?
A = A subfibre, B = B subfibre, C = dynein arms, D = radial spoke, E = pair of central
microtubules, F = plasma membrane, G = nexin crosslink.
Which of these i) contain phospholoipds - F
ii) consist of tubulin – A, B, E
iii) contain ATPase - C
iv) are responsible for converting microtubule sliding into cilium or
flagellum bending ? – D, G
Use the scale provided to estimate the diameter of a cilium axoneme.
0.25 
Fig XIV
Electron micrograph of tracheal ciliated epithelium
Gc is a mucous secreting goblet cell, Tb are tight junctions at the surface of the
epithelium between cells and Fi are actin filaments in the cellular cytoskeleton.
Identify cilia (C), microvilli(Vi), basal bodies (Bc), the plasma membranes of adjacent
cells(Cm) and mitochondria(Mi).
Why are so many mitochondria concentrated in the apical part of these ciliated cells?
Because of the energy needed for ciliary motility
Fig XV
Scanning electron micrograph of tracheal epithelium showing cilia(C) and microvilli
(M).
a.Using the scale provided, calculate the approximate length of the cilia seen.
Approximately 5-10 microns long
b.What do you think the pale round non-ciliated structures are?
Droplets of mucous
c. Kartagener’s disease is due to a defect in the dynein molecule found in cilia and
flagella which is partly responsible for the mechanism of movement. What do you think
would be some symptoms of the disease, considering where in the body cilia and flagella
are found?
Increased tendancy to respiratory tract infections due to poor tracheal ciliary function,
female infertility due to lack of fallopian tube ciliary function and male sterility due to
lack of spermatozoal flagellar function.
In about 50% of patients with this condition, a less easily understandable accompaniment
occurs – situs inversus – it seems that axonemal type motor proteins like Dynein are
important in left-right asymmetry in embryo formation.
Fig XVI
High power electron micrograph of a transverse section through the surface of a
particular epithelium.
What are these structures?
Microvilli
What is responsible for the stippling seen inside them?
Actin microfilaments
Where can they be found?
The intestinal epithelium in particular, but also on other epithelia, such as the bronchial
epithelium, to a lesser extent.
What is their function?
To increase the surface area of the cell, which is particularly important for absorption.
Using the scale provided, calculate their diameter and compare it to that of Cilia, (Fig
XIII)
Their diameter is about 0.1 microns, i.e. half that of cilia and flagella.
Fig XVII
Electron micrograph of microvilli seen in longitudinal section.
Using the scale provided, calculate the length of these structures and compare it to that of
the cilia calculated from Fig XIV.
1-2 microns long – that is smaller than cilia by a factor of 2-5
What does the core of the microvillus contain?
Bundles of actin microfilaments, laterally cross-linked to each other.
What is the fibrillar structure just below the microvillus.
The terminal web of actin microfilaments whichis continuous with the inside of the
microvilli.
Name one disease which is indirectly caused by a reduction in microvillus numbers.
Malabsorbtion is indirectly caused by a reduction in microvillus numbers, as can happen
in celiac disease.
Fig XVIII
This is an electron micrograph showing the junctional complex from intestinal
epithelium.
On the associated line diagram, name structures A to D, P and V.
A= tight junction ( zonula occludens), B = adherens junction (zonula adherens), C= spot
desmosome, D = desmosome plaque (made of desmogleins and desmoplakins), P =
plasma membrane, V = microvillus
What are the functions of A, B, C and V.
A – tight junctions provide a total closure of the intermembranous space, thus separating
the apical (luminal) surface of the cell from the baso-lateral compartment thus preventing
back diffusion of absorbed contents into the lumen, and entry of digestive enzumes into
the unprotected side of the cell.
B – zonula adherens - occurs as a whole belt around each cell in a tissue sheet,
effectively gluing that each cell to all its surrounding neighbours – it is an adhesive
junction.
C – desmosome – provides an added point of cell cell adhesion.
V – microvillus, increases absorbative surface.
What is the difference between the fibres which converge onto B and C.
The adherens junction is attached to actin microfilaments in the cell, whilst the
desmosome is attached to the intermediate tonofilaments.
What other differences are there between B and C.
B is continuous whilst C is a locallised point of cell-cell attachment, C has a thick
desmosome plaque deep to the membrane whilst B does not, the proteins involved in
each are different, with the catenins being primarily found in B whilst desmoplakin,
desmoglein and desmocollin are particularly found in C. Also their site of occurrence is
slightly different. Whilst B is particularly a feature of epithelia and occasionally neuronal
cells, C is particularly prominent in the heart muscle and the neck of the uterus.
How does the disease Pemphigus relate to C? Epidermolysis bullosa simplex which is a
blistering disease like Pemphigus, is caused due to mutation in one type of itermediate
filaments ( the keratins). Can you see a similarity in the mechanism??
Pemphigus is an auto-immune disease with auto-antibodies developing against some of
the desmosome proteins in the skin such as desmoglein or desmoplakin. This results in
skin blistering due to the prominent desmosomes in the skin and thie importance in
holding it together.
In Epidermolysis bullosa simplex, since the mutation in is skin intermediate filaments (
the keratins), these would be the filaments which link desmosomes to the two adjacent
cells, thus, this mutation can quite understandably give a similar pattern of disease.
Fig XIX
Cell showing meshwork of filaments stained with different immunofluorescent
antibodies.
Blue imunofluoresncence shows actin filaments
Green imunofluoresncence shows hollow microtubules
Pink imunofluoresncence shows vinculin – an actin-associated protein which helps
anchor the cell to matrix adhesion molecules such as Integrins.
What are the functions of the cell’s three types of cytoskeletal filaments - actin,
microtubules and intermediate fibres?
Actin filaments are involved in supporting the general skeleton of the cell, including the
terminal web and microvilli and has certain special functions in certain motile functions
of cells such as muscular contraction, in combination with myosin. It also links to the
zonula adherens, therefore being involved in intercellular adherence. It is also responsible
for cytoplasmic movements of most cell-types – such as chemotaxis, pseudopod
formation etc.
Microtubules are important in intracellular transport of components synthesised by the
synthetic machinery, such as transfer of vesicles from the Golgi apparatus to the cell
membrane. They are also responsible for mitotic spindles in cellular division, and also for
ciliary and flagellar movement. They are also responsible – for axonal transport.
Intermediate filaments help provide the structural support for the contractile machinery in
muscle cells especially smooth muscle cells. They also produce a nuclear scaffold which
helps shape the nucleus, they also provide intercellular mechanical support through
linking in to desmosomes.
Fig XX
Immunofluorescence staining of actin filaments in fibroblasts grown in tissue culture.
Fibroblasts are cells which are often resting in the body ( as in the top plate) but can
become motile with the right stimuli - for example migrating towards a wound to help in
wound healing- ( as in the bottom plate).
What differences can be noted in the shapes of the cells and the distribution of the actin
filaments?
The actin filaments are distributed around the cell in a relatively ordered manner in
various bundles crossing the cytoplasm in the top panel – ( showing an adherent cell). In
the lower panel, showing a cell moving with pseudopods being throw out, especially
towards the right, much of the actin mundles have become locallised to the periphery and
boundaries of the cell. Within the genral cytoplasm are much less visible finer sets of
fibres as opposed to clear bundles.
Fig XXI
Transmission electron micrograph of a pancreatic acinar cell. This is a typical protein
secreting cells which secretes digestive enzymes into the gastrointestinal tract.
Note the abundant Endoplasmic Reticulum(ER).
What are G and Z?
G is the Golgi apparatus, Z are zymogen granules – i.e. granules of pro-enzymes, ready to
be activatged by luminal contents and pH.
From the inset, which is the lumen of the cisterna and which is the space between two
adjacent cisternae? How can you distinguish them?
The lumen of the cisterna appears clear, whilst the space between them is cytoplasm,
filled with the dots which represent ribosomes on the cytoplasmic surface of the cisternae
membranes.
Which of these two is functionally continuous with the outside of the cell?
The lumen
How are the enzymes produced rendered safe, so as not to digest the cell from the inside?
As described above, by formation as pro-enzymes needing cleavage for activation, and
also by being packaged in compartments of membrane which on fusing to the cell
memebrane will release the enzymes outside the cell, through exocytosis.
Fig XXII
A high power scanning electron micrograph of the rough endoplasmic reticulum
What are the white bumps all over the RER?
Ribosomes
What surface is being indicated by the arrows? Would proteins on this surface be
glycosylated or not??
The surface indicated is the cytoplasmic (ribosome-covered) surface and the proteins
here would generally not be glycosylated.
Fig XXIII (This may have been missing from the set)
Transmission electron micrograph of a plasma cell – a lymphocyte activated to produce
antibodies.
What organellar structure occupies most of the cytoplasm?
Rough Endoplasmic reticulum
What protein modification would occur in the ER to the 4 peptide chains forming an
antibody?
They would be glycosylated with initial or core glycosylation, protein folding and the
catalysis of disulphide bond formation in order to link the 4 different peptide chains into
the structure of an antibody.
What differences would one expect between the cytoplasm and the nucleus of a resting
lymphocyte, and those of a plasma cell?
The cytoplasm of a resting lymphocyte is minimal, due to the resting state of the cell. In a
plasma cell, the cytoplasm is much increased, and full of RER required for the production
(ribosomes) and secretion (RER) of the antibodies. The nucleus of a resting lymphocyte
is almost completely heterochromatin and therefore very densely blue staining with H&E
stains, whilst that of a plasma cell is more euchromatic and less densely staining due to
the gene activity required to produce all the antibodies. The nuclear-cytoplasmic ratio
decreases during the switch from lymphocyte ( where it is very high) to plasma cell.
Can you identify the cytocentre?
Fig XXIV
Electron micrograph of a hepatocyte
Observe the SER and the dense clusters of glycogen granules in the intervening
cytoplasm. Some of the SER cisternae have dense granules within them – these are
lipoproteins.
What two functions of the SER do these features highlight?
Glycogen catalysis or breakdown, and lipoprotein synthesis
What other important functions does the SER perform in liver cells?
Detoxification and solubilisation, aiding secretion, membrane lipid synthesis
What important function does the SER perform in the gonads?
Cholesterol metabolism to create sex hormones.
Fig XXV
Electron micrograph of a cell following cryo-fracture showing the Golgi apparatus.
Note
the cis or forming face (GF)
Takes in vesicles incoming from RER and starts metabolising inherent proteins – enzyme
complexes include: enzymes involved in terminal glycosylation. Vesicles coated with
COPI proteins.
the trans or maturing face (GM)
Secretes or buds off vesicles as primary lysosomes and secretory vesicles : vesicles
coated with clathrin.
the intermediate lamellae (L)
Protein modification : enzymes include N-acetyl glucosamine transferease 1
several small and large vesicles (arrows)
secretory (exocytotic) vesicles and primary lysosomes.
Mention two cellular sites where these vesicles may be directed?
These may be the outer cell membrane (for exocytosis) and the lysosomal compartment.
(ie to fuse with phagocytic vesicles and digest their contents) They may also be directed
to other intracellular compartments to carry proteins required for those structures, using
the SNARE system.
Fig. XXVI
Electron micrograph showing stains identifying the cis and trans faces of the Golgi
Osmium tends to stain lipids by reacting with them– where are lipids synthesised. Does
it make sense that the highest concentrations of unmodified lipids are in the Cis Golgi?
Where are they coming from?
Yes it does make sense, since lipids are primarily systhesised on the RER, so high
concentrations of modified lipids would be found in the cis-Golgi which is made by
vesicles coming from the RER.
The trans Golgi is stained by an immuno-gold method for the enzyme acid phsophatase.
Where is this enzyme usually found in the cell?
Does this explain why the Trans Golgi is stained?
This enzyme is usually found in the lysosomes. These are budded off from the transGolgi – so again, this makes sense.
Fig XXVII
Electron micrograph stained histochemically for acid phosphatase.
Where would active acid phosphatase and other acid hydrolases be found?
In lysosomes.
How is the cell protected from their digestive action?
The eyzymes within a lysosome are all optimally catalytic as active enzymes at an acidpH of 5.0 or so. Thus, after being isolated in a protective sphere of lipid membrane
which is also heavily glycosylated, the pH within that compartment is changed by proton
pumps and the enzymes, now isolated, are activated, to be fused to phagosomes, again
isolated from the cellular components.
Fig XXVIII
Electron micrograph of lysosomes.
Why are lysosomes so heterogenous in size and in texture/staining?
This is due to the fact that they are produced by fusion of endosomes and will later fuse
with autophagosomes of internal organelles requiring degradation, or heterophagosomes
of phagocytosed material from outside the cell. These seconday components obviously
change their structure and staining.
What is their function?
To digest and recycle components of the cell and also anything imported from the
outside.
If they are so heterogenous in shape, what identifies them?
Their internal acid pH and also their enzyme content.
Hurler’s disease is due to a mutation in a lysosomal enzyme and is one of many storage
disorders. What would you expect to see on electron microscopy of such cells?
One would expect to see numerous inclusion particles of undigested material.
Fig XXIX
Electron micrograph of various spherical organelles.
Apart from peroxisomes and lysosomes, what are the other organelles.
Mitochondria
How would this organelle differ from the other two in its plasma membranes?
It is surrounded by a double layer of membranes with an inter-membrane space unlike
lysosomes and peroxisomes which are only surrounded by a single unit membrane.
What is the function of peroxisomes?
They contain numerous enzymes which act to control oxygen free radicals, and peroxide
which is often generated within the peroxisomes themselves. For this reason they have a
large store of the enzyme catalase which breaks down H2O2They also have a role in the
breakdown of long chain fatty acids, and in the detoxification of certain molecules.
Fig XXX
Transmission electron micrograph of a pancreatic acinar cell.
Identify the organelles A, and D and the large sausage shaped organelle in the centre.
A = Peroxisome, D = RER, long organelle = Mitochondrion
Also identify the cellular and organellar components – B, C and E.
B= cristae, C = cation granules, E = ribosomes
On the associated diagram, name compartments/spaces or membranes 1 to 5.
1= inner mitochondrial membrane, 2 = intermembranous space, 3 = crista, 4=
intramembranous space, 5= outer mitochondrial membrane.
In which of these would you find the a)Matrix, b) electron transport chain, c) cardiolipin
synthetic enzymes, d) ATPsynthase, e) DNA, f) ribosomes, g) Krebbs cycle enzymes?
a in 4; b in 1,3; c in 5; d in 2; e in 4; f in 4; g in 4.
Fig XXXI
Electron micrograph.
How many cells are seen in this view?
6
Take note of the organelles in their cytoplasm and the state of the chromatin in their
nucleus.
From these observations, what can be inferred about the function and activity of these
cells?
They are highly active cells(much euchromatin, little heterochromatin, many
mitochondria) involved in protein synthesis(much RER) and possibly also in lipid
metabolism and or detoxofication (some SER).
Fig XXXII
This is an electron micrograph of an endocrine gland.
Note the cytoplasmic organelles – including the numerous small vesicles in the cytoplasm
(paper tag B) and the strange looking organelles which are very abundant (paper tag A,
also * and M). PM is plasma membrane, CT is connective tissue, EN are endothelial
cells and P is a pore in the endothelium
How many cells can one see part of ( exclude the very edges and corners of the picture)?
7 or 8 – parts of 4 endocrine cells, 2-3 endothelial cells, and part of a red blood cell (
dense structure inside the capillary)
Can you identify the organelles labelled A and B?
A are mitochondria and B are SER cisternae.
What is different in these organelles from those found in other cells? Why?
The mitochondria here have tubular cristae unlike the more common shelf-like cristae
found in other cell types.
What are the special function of these organelles in this tissue?
They are involved with steroid synthesis and the energy required for this process.