Basic Principles of Medicine 1

Module: Foundation to Medicine

Lecture No: 4

Cellular Organization II

E. Koppelman, Ph.D.
St Georges University ©
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 Assigned Reading

Histology – A Text and Atlas

Pawlina 7th Edition
Chapter 2: Cell Cytoplasm Pg. 55-73
http://meded.lwwhealthlibrary.com/book.aspx?bookid=1316

Lippincott's Illustrated Reviews: Cell & Molecular Biology

Chandler & Viselli 2010
Chapter 4: Cytoskeleton Pg. 35-46

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Objectives
List the three major types of protein filaments that form the cytoskeleton.
Identify & describe the structure, function and assembly of microtubules,
intermediate filaments and microfilaments.
Discuss the compounds that affect microtubules.
Discuss the compounds that affect microfilaments.
Describe the structure and function of the centrosome.
Identify & describe the structure and function of primary cilia, motile cilia and
flagella.
List and describe the molecular motor proteins associated with microfilaments and
microtubules.
Describe role of the cytoskeleton in intracellular transport and cellular motility.
Describe cell motility across a substrate.
Describe the three types of membrane protrusion structures.
Describe the role of actin polymerization in membrane protrusion and motility.
Describe the types of intracellular inclusions.
Cytoskeleton

Intermediate
Actin Microtubules Filaments

• 3 major components • Functions

1. Actin filaments (microfilaments)
• Structural support & stability
• Actin
• 7 nm diameter • Organization
2. Intermediate filaments • Cell division
• 6 classes, numerous proteins
• 8-12 nm diameter • Cell movement
3. Microtubules • Tracks for motor proteins to move
• Tubulin
Cytoskeleton
• 25 nm diameter
organelles & vesicles within cells
 Microtubules
• Functions
• Intracellular transport
• Movement of vesicles & organelles via motor
proteins

• Cell motility
• Movement of cilia and flagella via motor
proteins
• Cell elongation and movement

• Mitotic Spindle
• Attachment of chromosomes & their movement
during cell division

• Structure • Rigid intracellular skeleton

• Non-branching, rigid, hollow tubes • Maintenance of cell shape & polarity
• α and β tubulin protein subunits
• Polar
• Minus (-) end • Assemble and disassemble as the needs of the
cell change
• Plus (+) end
Centrioles

• Structure
• 9 triplets of microtubules arranged around a central axis
• Each triplet consists of 1 complete and 2 incomplete microtubules fused

• Functions
• Organize the centrosome
• Basal body formation
• Provide basal bodies necessary for assembly of cilia and flagella
• Mitotic spindle formation
• Formation of centrosome & alignment of the mitotic spindle during cell division
Centrosome
• Microtubule Organizing Center (MTOC)

• Structure
• Contains a pair of centrioles
• Arranged such that one is perpendicular to the
other

• Amorphous protein matrix

• >200 proteins

• Gamma-tubulin ring complexes

• Nucleation sites for microtubules

• Function
• Organize microtubules
• Initiate microtubule formation
• Microtubules are nucleated at the centrosome at
their negative (-) ends
• Positive (+) ends point out and grow toward the
cell periphery
Microtubules
Polymerization
• Organized & directed by microtubule organizing centers
• Basal bodies
• Centrosome

• GTP dependent

• Highly dynamic (mitotic spindle) or relatively stable (cilia)

• Change in length
• Fast growth at positive (+) end
• Slow growth or disassembly at negative (-) end

• Microtubule associated proteins (MAPs)

• Tau proteins
• Abundant in neurons of the CNS
• Stabilize axonal microtubules
• Hyperphosphorylation of tau proteins can result in self-assembly
of tangles
• Linked to Alzheimer’s disease
Microtubules

• Centrosome & Microtubules

• EM x30,000
• Centrioles (C)
• Nucleus (N)
• Microtubules (MT)
• Rough endoplasmic reticulum (rER)
• Golgi (G)
• Mitochondria M
Mitotic Spindle Poisons

• Colchicine
• Anticancer compound
• Prevents polymerization
• Binds to unpolymerized tubulin molecules
• If given to dividing cells, mitotic spindle
breaks down
• Programmed cell death
• Related compounds
• Vinca alkaloids
• Taxol® (Paclitaxel)
• Vinblastine • Anticancer compound
• Vincristine • Stabilizes and prevents microtubule
disassembly
• Preferentially binds tubulin within assembled
microtubules
• Arrests dividing cells in mitosis
• Unable to achieve metaphase spindle conformation
• Programmed cell death
 Microtubule Motors

1. Dynein Family
• Move along microtubules toward the (-) end

• 2 members
1. Cytoplasmic dyneins 1.
2. Axonemal dynein
• Located in Cilia & Flagella

• Binding sites for vesicles, organelles or another

microtubule

• Largest & fastest of the known molecular

motors
• Dynein 14 um/sec vs. Kinesin 3 um/sec 2.
Microtubule Motors

2. Kinesin Family
• Move along microtubules toward the (+) end
• Binding sites for vesicles, organelles,
microtubules
• ~ 40 distinct kinesins in humans
Cilia & Flagella

• Motile structures
• Highly specialized

• Microtubules and axonemal dynein motor

proteins
• Characteristic arrangement found in almost all
eukaryotic flagella and cilia

• Movement produced by the bending of the

core (axoneme)

• Accessory proteins cross-link adjacent

microtubules together
• As a result, dynein motors produce a bending
movement
Cilia

• Microtubule-based, hair-like structure

• Motile
• Beat in synchronous pattern
• 9 + 2 microtubule arrangement
• Pair of Dynein arms
• Motor protein
• Binds adjacent microtubule

• Move fluid and particles along epithelial

surfaces

• Anchored to cell via the basal body

• Thin, dark-staining band at base of cilia
Primary Cilia
• Sensory antennae
• Photoreceptors
• Outer segment of rods derived from primary cilia

• Chemoreceptors
• Odor detection by receptors on primary cilia of
olfactory neurons

• Mechanoreceptors
• Primary cilia of epithelial cells monitors the flow of
• Microtubule-based, antennae-like structure fluid through the kidney tubules
• Defects underlie a variety of disorders
• 9 + 0 arrangement
• Polycystic Kidney Disease

• Emanates from almost all cells

• Anchored to cell via the basal body

• Develops from one centriole following cell division

Intermediate Filaments
• Rope-like filaments

• Structure
• Formed from non-polar & highly
variable subunits

• Functions
• Structural
• Stabilize cell structure
• Mechanical strength
• Maintain the position of the nucleus
and other organelles
• Resist shearing forces
• Extend across cytoplasm
• Connecting with desmosomes &
hemidesmosomes

• Essential for integrity of cell-cell &

cell-ECM junctions
6 Classes Intermediate Filaments
• 1 & 2: Keratins
• Acid & basic cytokeratins
• Diverse group
• >50 isoforms
• Found in all epithelial cells

• 3: Vimentin & Vimentin-like

• Diverse family
• Most widely distributed in the body
• Vimentin
• Most abundant in mesoderm-derived cells
• Vimentin-like found in a variety cells
• Desmin: muscle cells
• Glial fibrillary acid protein (GFAP): glial cells & astrocytes

• 4: Neurofilaments
• Assembled from neurofilament proteins of different molecular weights
• Extend from cell body into the ends of axons & dendrites
• Provide structural support
• Found primarily in neurons

• 5: Lamins
• Found in nucleus of all nucleated cells
• Nuclear lamina
• Lamin A & B proteins

• 6: Beaded Filaments
• Eye lens-specific group
Actin Filaments (Microfilaments)
• Made of the protein actin
• G-actin
• Free actin molecules in the cytoplasm
• F-actin
• Polymerized actin in a filament
• ATP dependent

• Polarized structures
• Fast growing (+) positive end
• Slow growing (-) negative end

• May exist as single filaments, in bundles, or

in networks

• Variety of cell functions

• Anchorage
• Structural core of microvilli & stereocilia
• Cell motility
• Locomotion
• Extension of cell processes
Actin & Fungal Toxins

• Phalloidin
• Toxin found in Amanita phalloides
• Used in cytoskeleton research
• Disrupts normal function of actin
• Binds F-actin more tightly and G-actin
• Promotes excessive polymerization and inhibits
depolymerization
• Inhibits cell movement
• Other toxins (amatoxins) are responsible for toxic
effects following oral ingestion
• Liver and kidney failure & death 4-8 days after consumption

Amanita phalloides -F. Stevens

• Cytochalasins “death caps” or “angels of death”
• Other fungal products
• Block polymerization of actin
• Can be used to inhibit cell movement, division &
induce programmed cell death
Microvilli

• Cylindrical, membrane-bound cytoplasmic projections

• 1-3 µm in length

• Core of 25-30 actin microfilaments

• Crosslinked by villin
• Anchored into the terminal web
Stereocilia

• Unusually long microvilli

• Up to 120 µm in length

• Actin filament bundle

anchored to terminal web

• Limited distribution
• Epididymis
• Proximal ductus deferens
• Sensory hair cells of inner ear
 Actin Motors
Myosin Family
• Human genome includes ~40 different myosin
genes
• Myosin II
• Generates the force for skeletal muscle
contraction
• Formed from 2 heavy chains & 4 light chains
• Tail-tail interactions result in formation of
bipolar thick filaments
• Several hundred myosin heads
• Each head binds and hydrolyzes ATP
Actin Motors

Myosin Structural Changes

• Stage 1: Attachment
• Rigor conformation
• Stage 2: Release
• ATP binds
• Reduces myosin affinity for actin
• Stage 3: Bending
• ATP hydrolysis
• Conformational change
• Stage 4: Force generation
• Weak binding of myosin to actin causes release
of inorganic phosphate
• Release triggers tight binding & power-stroke
• Force-generating conformational change
• Stage 5: Reattachment
• Rigor conformation
 Cell Movement

Crawling Neutrophil Chasing a Bacterium

https://www.youtube.com/watch?v=I_xh-bkiv_c
Cell Movement

Complex & integrated process

• Protrusion
• Actin polymerization at plus (+) end
protrudes lamellipodium

• Attachment
• Focal adhesions anchor the actin
cytoskeleton to the extracellular matrix
via integrin proteins

• Contraction
• Bulk of the trailing cell & cytoplasm is
drawn forward
Cell Movement
• Plasma membrane protrusions
• Driven by actin polymerization

• Different types of protrusion structures

based on organization of actin
• Filopodia
• Finger-like projections
• Core of long, bundled actin filaments
• Lamellipodia
• Sheet-like structures
• Pseudopodia
• 3 dimensional projections
Cell Movement

Actin Polymerization
• Actin filaments mostly oriented with (+)
end facing forward

• (-) ends frequently attached to sides of

other actin filaments via actin-related
protein (ARP) complexes

• Actin web as a whole undergoes

treadmilling
• Assembling at the front
• Disassembling at the rear
Cell Movement
Actin Polymerization
• ARP complex highly concentrated near front of lamellipodia
where actin nucleation most active
• Actin filaments (red)
• ARP complex (green)

• Dense actin filament meshwork at leading edge

 Neutrophil Migration

Extravasation
1. Rolling
2. Activation
3. Adhesion
Chemotaxis
4. Transendothelial migration • Movement within the tissue along a chemotactic
• Process of diapedesis gradient towards the source of inflammation
• Extension of a pseudopod between endothelial cells • N-formylated peptides
• Pass through the basement membrane into the tissue • Peptides attached to extracellular matrix
Neutrophil Migration
 Inclusions
• Cytoplasmic or nuclear structures formed • Glycogen
from metabolic products of the cell • Non-membrane-bound, TEM dense bodies
• Single (beta) 20-30 nm particles or rosettes (alpha)
• Storage form of glucose
• Pigments • Catabolism releases glucose for energy
• Membrane-bound
• Lipid
1. Lipofuscin • Non-membrane bound, TEM dense
• Brownish-gold pigment
• Generally seen in non-dividing cells • Fat droplets
• Accumulates over years • Spherical droplets of triglyceride
• “Wear & tear” pigment • Liquid at body temperature
• Conglomerate of lipids, metals, organic molecules • Energy store and source of short carbon chains
for membrane synthesis
2. Hemosiderin • Lipid storage diseases (lipidoses)
• Brown pigment • Lipid droplets accumulate in abnormal amounts or
• Iron-storage complex found in the cytoplasm locations
• Likely formed by indigestible residues of
hemoglobin
• Easily demonstrated in the spleen

3. Melanin
• Brown pigment
Glycogen

EM x10,000 EM x52,000
Lipid
a. H & E x320
b. Osmium tetroxide x320
c. EM x24,000
a. Lipofuscin b. Melanin
• H & E x320 • Modified Azan x600
• Sympathetic ganglion cells • Substantia nigra nerve cells