This document discusses several cytoplasmic inclusions including endosomes, peroxisomes, proteasomes, and other inclusions. It describes the structure and functions of endosomes, including their role in sorting and transporting materials within cells. Peroxisomes contain oxidative enzymes and are involved in processes like breaking down hydrogen peroxide. Proteasomes are complexes responsible for degrading malformed and tagged proteins. Other inclusions discussed include lipofuscin, hemosiderin, glycogen, and lipid droplets.

1. Endosomes.Peroxisomes. Proteasomes
Cytoplasmic inclusions
Structure.Functions.Their role in
protection of cell
10/12/2017nihal yuzbasheva

2. Endosomes
• Endosomes are divided into two compartments: early endosomes, near the
periphery of the cell, and late endosomes, situated deeper within the
cytoplasm near nucleus and Golgi apparatus.
• Early endosomes, are restricted to a portion of the cytoplasm near the cell
membrane where vesicles originating from the cell membrane fuse.
Early endosomes in live HeLa
cells identified after a 10-minute
incubation with green
10/12/2017nihal yuzbasheva

3. • From here, many vesicles return to the plasma membrane. However, large
numbers of vesicles originating in early endosomes travel to deeper
structures in the cytoplasm called late endosomes. The latter typically
mature into lysosomes.
Electron micrograph of an early endosome.
This deep-etch electron micrograph shows the structure of an
early endosome in Dictyostelium. Early endosomes are located
near the plasma membrane and, as in many other sorting
compartments, have a typical tubulovesicle structure. The tubular
portions contain the majority of integral membrane proteins
destined for membrane recycling, whereas the luminal portions
collect secretory cargo proteins. The lumen of the endosome is
subdivided into multiple compartments, or cisternae, by the
invagination of its membrane and undergoes frequent changes in
shape. 15,000. (Courtesy of Dr. John E. Heuser, Washington
University School of Medicine.)
Endosomes can be viewed either as stable cytoplasmic organelles or as
transient structures formed as the result of endocytosis.
10/12/2017nihal yuzbasheva

4. • Recent experimental observations of endocytotic pathwaysconducted in
vitro and in vivo suggest two different models that explain the origin and
formation of the endosomal compartments in the cell:
• The stable compartment model describes early and late endosomes as
stable cellular organelles connected by vesicular transport with the external
environment of the cell and with the Golgi apparatus. Coated vesicles
formed at the plasma membrane fuse only with early endosomes because of
their expression of specific surface receptors. The receptor remains a
resident component of the early endosomal membrane.
• In the maturation model, early endosomes are formed de novo from
endocytotic vesicles originating from the plasma membrane. Therefore, the
composition of the early endosomal membrane changes progressively as
some components are recycled between the cell surface and the Golgi
apparatus. This maturation process leads to formation of late endosomes
and then to lysosomes. Specific receptors present on early endosomes (e.g.,
for coated vesicles) are removed by recycling, degradation, or inactivation
as this compartment matures.
10/12/2017nihal yuzbasheva

5. • Endosomes destined to become lysosomes receive newly synthesized
lysosomal enzymes that are targeted via the mannose-6-phosphate
receptor.
• Some endosomes also communicate with the vesicular transport system of
the rER. This pathway provides constant delivery of newly synthesized
lysosomal enzymes, or hydrolases.
• A hydrolase is synthesized in the rER as an enzymatically inactive precursor
called a prohydrolase. This heavily glycosylated protein then folds in a
specific way so that a signal patch is formed and exposed on its surface.
• The signal patch on a protein destined for a lysosome is then modified by
several enzymes that attach mannose-6-phosphate (M-6-P) to the
prohydrolase surface. M-6-P acts as a target for proteins possessing an M-
6-P receptor.
10/12/2017nihal yuzbasheva

6. 10/12/2017nihal yuzbasheva
The Mannose-6-Phosphate Pathway

7. 10/12/2017nihal yuzbasheva
• Early and late endosomes differ in their cellular localization,
morphology, and state of acidification and function.
An early endosome has a tubulovesicular
structure: The lumen is subdivided into
cisternae that are separated by
invagination of its membrane. It exhibits
only a slightly more acidic environment
(pH 6.2 to 6.5) than the cytoplasm of the
cell.
In contrast, late endosomes have a more
complex structure and often exhibit
onionlike internal membranes. Their pH is
more acidic, averaging 5.5.

8. • TEM studies reveal specific vesicles that transport substances between early
and late endosomes. These vesicles, called multivesicular bodies (MVBs),
are highly selective transporters.
• Because late endosomes mature into lysosomes, they are also called
prelysosomes
10/12/2017nihal yuzbasheva
Pathways for delivery of newly synthesized
lysosomal enzymes. Lysosomal enzymes (such as
lysosomal hydrolases)
are synthesized and glycosylated within the rough endoplasmic
reticulum (rER). The enzymes then fold in a specific
way so that a signal patch is formed, which allows for further
modification by the addition of M-6-P, which allows the enzyme to
be targeted to specific proteins that possess M-6-P receptor
activity.
M-6-P receptors are present in the TGN of the Golgi apparatus,
where the lysosomal enzymes are sorted and packaged
into vesicles later transported to the early or late endosomes.

9. 10/12/2017nihal yuzbasheva
• The major function of early endosomes is to sort and recycle proteins
internalized by endocytotic pathways.
• The morphologic shape and geometry of the tubules and vesicles emerging
from the early endosome create an environment in which localized changes
in pH constitute the basis of the sorting mechanism.
• This mechanism includes dissociation of ligands from their receptor
protein; thus, in the past, early endosomes were referred to as
compartments of uncoupling receptors and ligands (CURLs).
Following endocytosis, ligand–drug
conjugates may be trafficked through
different intracellular compartments,
depending on the receptor that is exploited
for the internalization of the conjugate.
Some of the more common compartments
that are encountered during intracellular
trafficking include: early endosomes;
compartments for uncoupling of receptor
and ligand (CURLs), where dissociation of
the conjugate from the receptor may occur;
recycling endosomes, which can deliver the
internalized receptor back to the cell surface;
and lysosomes, where the receptor and the
conjugate can be degraded.

10. 10/12/2017nihal yuzbasheva
• In addition, the narrow diameter of the tubules and vesicles may also aid in
the sorting of large molecules, which can be mechanically prevented from
entering specific sorting compartments. After sorting, most of the protein is
rapidly recycled, and the excess membrane is returned to the plasma
membrane.
• The fate of the internalized ligand–receptor complex depends on the
sorting and recycling ability of the early endosome.
• The following pathways for processing internalized ligand–receptor
complexes are present in the cell:The receptor is recycled and the ligand is
degraded, Both receptor and ligand are recycled,Both receptor and ligand
are degraded, Both receptor and ligand are transported through the cell.

11. • The receptor is recycled and the ligand is degraded.Surface receptors allow the
cell to bring in substances selectively through the process of endocytosis. This
pathway occurs most often in the cell; it is important because it allows surface
receptors to be recycled. Most ligand–receptor complexes dissociate in the
acidic pH of the early endosome. The receptor, most likely an integral
membrane protein , is recycled to the surface via vesicles that bud off the ends of
narrow-diameter tubules of the early endosome. Ligands are usually
sequestered in the spherical vacuolar part of the endosome that will later form
MVBs, which will transport the ligand to late endosomes for further degradation
in the lysosome . This pathway is described for the low-density lipoprotein
(LDL)–receptor complex, insulin–glucose transporter (GLUT) receptor
complex, and a variety of peptide hormones and their receptors.
• Both receptor and ligand are recycled. Ligand–receptor complex dissociation
does not always accompany receptor recycling. For example, the low pH of the
endosome dissociates iron from the iron-carrier protein transferrin, but
transferrin remains associated with its receptor. Once the transferrin–receptor
complex returns to the cell surface, however, transferrin is released. At neutral
extracellular pH, transferrin must again bind iron to be recognized by and
bound to its receptor. A similar pathway is recognized for major
histocompatibility complex (MHC) I and II molecules, which are recycled to the
cell surface with a foreign antigen protein attached to them.
10/12/2017nihal yuzbasheva

12. • Both receptor and ligand are degraded. This pathway has been identified for
epidermal growth factor (EGF) and its receptor. Like many other proteins, EGF binds
to its receptor on the cell surface. The complex is internalized and carried to the early
endosomes. Here EGF dissociates from its receptor, and both are sorted, packaged in
separate MVBs, and transferred to the late endosome. From there, both ligand and
receptor are transferred to lysosomes, where they are degraded
• Both receptor and ligand are transported through the cell. This pathway is used for
secretion of immunoglobulins (secretory IgA) into the saliva and human milk. During
this process, commonly referred to as transcytosis, substances can be altered as they
are transported across the epithelial cell. Transport of maternal IgG across the
placental barrier into the fetus also follows a similar pathway.
10/12/2017nihal yuzbasheva

13. Peroxisomes (Microbodies)
• Peroxisomes (microbodies) are small (0.5 m in diameter), membrane-
limited spherical organelles that contain oxidative enzymes, particularly
catalase and other peroxidases. Virtually all oxidative enzymes produce
hydrogen peroxide (H2O2) as a product of the oxidation reaction.
10/12/2017nihal yuzbasheva

14. • The catalase universally present in peroxisomes carefully regulates the
cellular hydrogen peroxide content by breaking down hydrogen peroxide,
thus protecting the cell.
• In addition, peroxisomes contain D-amino acid oxidases,β -oxidation
enzymes, and numerous other enzymes.
• Peroxisomes in hepatocytes are responsible for detoxification of ingested
alcohol by converting it to acetaldehyde.
10/12/2017nihal yuzbasheva
The -oxidation of fatty acids is also a
major function of peroxisomes.
A protein destined for peroxisomes must
have a peroxisomal targeting signal
attached to its carboxy-terminus.
In most animals, but not humans,
peroxisomes also contain urate oxidase
(uricase), which often appears as a
characteristic crystalloid inclusion
(nucleoid).

15. 10/12/2017nihal yuzbasheva
• In the most common inherited disease related to nonfunctional
peroxisomes, Zellweger syndrome, which leads to early death, peroxisomes
lose their ability to function because of a lack of necessary enzymes. The
disorder is caused by a mutation in the gene encoding the receptor for the
peroxisome targeting signal that does not recognize the signal Ser-Lys-Leu
at the carboxyterminus of enzymes directed to peroxisomes.
Contribution of Fetal MR
Imaging in the Prenatal
Diagnosis of Zellweger
Syndrome

16. Proteasomes
• Proteasomes are small organelles composed of protein complexes that are
responsible for proteolysis of malformed and ubiquitin-tagged proteins.
• The protein population of a cell is in a constant flux as a result of the
continuous synthesis, export, and degradation of these macromolecules.
10/12/2017nihal yuzbasheva
Frequently, proteins, such as those that act in
metabolic regulation, have to be degraded to
ensure that the metabolic response to a single
stimulus is not prolonged.

17. • Additionally, proteins that have been denatured, damaged, or malformed
have to be eliminated; moreover, antigenic proteins that have been
endocytosed by antigen-presenting cells (APCs) have to be cleaved into
small polypeptide fragments (epitopes) so that they can be presented to T
lymphocytes for recognition and the mounting of an immune response.
• The process of cytosolic proteolysis is carefully controlled by the cell, and it
requires that the protein be recognized as a potential candidate for
degradation. This recognition involves ubiquination, a process whereby
several ubiquitin molecules (a 76-amino acid long polypeptide chain) are
attached to a lysine residue of the candidate protein to form a
polyubiquinated protein.
10/12/2017nihal yuzbasheva

18. 10/12/2017nihal yuzbasheva
• Once a protein has been thus tagged, it is degraded by proteasomes,
multisubunit protein complexes that have a molecular weight in excess of 2
million daltons.
• During proteolysis, the ubiquitin molecules are released and reenter the
cytosolic pool. The mechanism of ubiquitination requires:
1.The cooperation of a series of enzymes, including ubiquitin-activating
enzyme
2.A family of ubiquitin-conjugating enzymes
3.A number of ubiquitin ligases each of which recognizes one or more
substrate proteins

19. INCLUSIONS
• Inclusions are cytoplasmic or nuclear structures with characteristic staining
properties that are formed from the metabolic products of cell. They are
considered nonmoving and nonliving components of the cell.
• Some of them, such as pigment granules, are surrounded by a plasma
membrane; others (e.g., lipid droplets or glycogen) instead reside within the
cytoplasmic or nuclear matrix.
10/12/2017nihal yuzbasheva

20. • Lipofuscin is a brownish-gold pigment visible in routine H&E preparation.
It is easily seen in nondividing cells such as neurons and skeletal and
cardiac muscle cells.
• Lipofuscin accumulates during the years in most eukaryotic cells as a result
of cellular senescence (aging); thus, it is often called the “wear-and-tear”
pigment.
• Lipofuscin is a conglomerate of oxidized lipids, phospholipids, metals, and
organic molecules that accumulate within the cells as a result of oxidative
degradation of mitochondria and lysosomal digestion.
10/12/2017nihal yuzbasheva
Phagocytotic cells such as
macrophages may also contain
lipofuscin, which accumulates from
the digestion of bacteria, foreign
particles, dead cells, and their own
organelles. Recent experiments
indicate that lipofuscin
accumulation may be an accurate
indicator of cellular stress.

21. • Hemosiderin is an iron-storage complex found within the cytoplasm of
many cells. It is most likely formed by the indigestible residues of
hemoglobin, and its presence is related to phagocytosis of red blood cells.
• Hemosiderin is most easily demonstrated in the spleen, where aged
erythrocytes are phagocytosed, but it can also be found in alveolar
macrophages in the lung tissue, especially after pulmonary infection
accompanied by small hemorrhage into the alveoli.
10/12/2017nihal yuzbasheva
Hemosiderin-laden macrophages in the lung.
KU Collection
It is visible in light microscopy as
a deep brown granule, more or
less indistinguishable from
lipofuscin.
Hemosiderin granules can be
differentially stained
using histochemical methods for
iron detection.

22. • Glycogen is a highly branched polymer used as a storage material for
glucose. It is not stained in the routine H&E preparation. However, it may
be seen in the light microscope with special fixation and staining
procedures (such as toluidine blue or the PAS method).
• Liver and striated muscle cells, which usually contain large amounts of
glycogen, may display unstained regions where glycogen is located.
Glycogen appears in EM as granules 25 to 30 nm in diameter or as clusters
of granules that often occupy significant portions of the cytoplasm
10/12/2017nihal yuzbasheva

23. • Lipid inclusions (fat droplets) are usually nutritive inclusions that provide
energy for cellular metabolism. The lipid droplets may appear in a cell for a
brief time (e.g., in intestinal absorptive cells) or may reside for a long period
(e.g., in adipocytes)
• In adipocytes, lipid inclusions often constitute most of the cytoplasmic
volume, compressing the other formed organelles into a thin rim at the
margin of the cell. Lipid droplets are usually extracted by the organic solvents
used to prepare tissues for both light and electron microscopy. What is seen as
a fat droplet in light microscopy is actually a hole in the cytoplasm that
represents the site from which the lipid was extracted.
10/12/2017nihal yuzbasheva
In individuals with genetic
defects
of enzymes involved in lipid
metabolism, lipid droplets
may accumulate in abnormal
locations or in abnormal
amounts. Such diseases are
classified as lipid storage
diseases.

24. • Crystalline inclusions contained in certain cells are recognized in the light
microscope. In humans, such inclusions are found in the Sertoli
(sustentacular) and Leydig (interstitial) cells of the testis.
• With the TEM, crystalline inclusions have been found in many cell types
and in virtually all parts of the cell, including the nucleus and most
cytoplasmic organelles.
• Although some of these inclusions contain viral proteins, storage material,
or cellular metabolites, the significance of others is not clear.
10/12/2017nihal yuzbasheva
Schaumann bodies with crystalline inclusions,
polarized
Multinucleate plasma cell with intracytoplasmic
crystalline inclusions, (Geimsa stain, ×1000)