Cytoskeletal proteins & inclusions

Learning objectives

protein filaments

• dynamic, internal “skeleton”

• three primary types

  • actin microfilaments

  • intermediate filaments

  • microtubules

• differ in size & function

• hundreds of cytoskeleton-associated proteins regulate distribution & behavior of cytoskeletal proteins

Actin filaments

• Thinnest component of cytoskeleton

• Made of globular actin monomers 

  • Each with ATP binding site

• Polymerize into a  microfilament (6-8 nm)

  • Two strings of beads twisted together

  • Monomers oriented in one direction producing polarity

  • “Plus end” and a “minus” end

• Dynamic structures

  • “Plus” end is fast growing

  • “Minus” end is slow growing

Microtubules

• hollow, non-branching cylinders

• Heterodimer made of alternating globular tubulin molecules

• Alpha tubulin at “minus” end

• Beta tubulin at “plus” end

  • Each beta-tubulin globule is bound to GTP

• Each microtubule made of 13 parallel protofilaments

• §Dynamic structures

  • “Plus” end is growing

  • “Minus” end is non-growing

nucleation site

Both actin filaments and microtubules grow from a _______ _____

Nucleation site

• a cellular location or protein complex where the formation of new filaments, like actin and microtubules, is initiated.

  • (2 monomers bind weakly; 3 form a more stable group)

• Binding of subunits causes hydrolysis of ATP (actin) or GTP (microtubules), which decreases strength of binding – dynamic instability

  • ADP/GDP molecule dissociates from minus end

Where is actin’s nucleation site most often found?

plasma membrane

Where are microtubules’ nucleation site most often found?

Microtubule organizing centers (MOC)

cytosolic proteins

_____ _____ can stabilize or destabilize actin filaments & microtubules

• Some bind actin monomers or tubulin dimers

  • Prevent binding to filaments/tubules

  • Maintains reserve pool of monomers

• Capping protein – stabilizes plus end of filament

• Another protein severs the filament

Bundling proteins

_____ _____ can provide stronger, stable actin structures

• some promote nucleation at plasma membrane

• some cross-link into parallel arrays

• some bundle actin filaments at an angle to produce a web-like network

nucleus

Shape and number of actin filaments and microtubules is regulated by the ____ by altering the number of regulatory proteins

What are the functions of actin filament?

• Maintains cell shape & anchors membrane proteins

  • Can provide either flexible or stable support

• Motility

  • Scaffold for myosin (motor protein) in muscle cells

  • Cellular locomotion of other cells

• Movement of vesicles

• Cytokinesis after completion of mitosis

  • Myosin needed for contraction of ring

Actin bundles

___ _____ in core of microvilli help them to remain upright

• Microvilli are cell membrane projections that increase surface area

  • Particularly well developed in cells that absorb things

– Intestine

– Kidney

• Microvilli are supported by stable bundles of actin filaments

• Actin bundles are anchored to terminal web of actin at base of microvilli

Microvilli

• cell membrane projections that increase surface area

  • Particularly well developed in cells that absorb things

– Intestine

– Kidney

• are supported by stable bundles of actin filaments

myosin

____ produces cellular contraction by sliding actin filaments in opposite directions

• Several types of myosin motor proteins

  • 1-2 heads & a tail

• Head repetitively binds and releases actin filament in a swinging motion, moving down filament

  • Hydrolyze ATP in process

• Myosin can pull a vesicle along an actin filament

• Myosin pulls 2 actin filaments toward each other to     facilitate cell movement

  • 1 anchored to back of cell

  • 1 anchored further forward in cell

What are the functions of microtubules?

• Functions

  • Cilia and flagella

  • Mitotic spindle

  • Cytostructural support – anchor organelles

  • Motor proteins (dynein and kinesin) move vesicles along microtubular “railroad tracks”

• Some chemotherapy drugs (e.g. vinblastine) suppress microtubule dynamics

Microtubule Organizing Centers (MTOC)

• Sites that localize microtubule minus ends

  • Microtubule nucleation (g-tubulin)

  • Stabilization & anchoring

  • Arrangement within cell

• Arrangement of microtubules varies with cell type

Centrosome

• the best studied MTOC (microtubule organizing center)

• Used by all cells for generating the mitotic spindle during mitosis and meiosis

• During mitosis, duplicated centrosomes serve as poles for mitotic spindle

• Centriole contains 2 centrioles surrounded by a matrix of proteins

  • Short, rod-like, cylinders each built from 9 microtubule triplets.

  • Function is a mystery

Microtubules

Cilia (and flagella) are composed of ______

• 9 pairs of circularly arranged microtubule doublets

• 2 central microtubules (9+2)

• Linker proteins

• Arise from basal body (nucleation site)

Movement along microtubules are mediated by which motor proteins?

Dynein and kinesin

• two globular ATP-binding motor heads and a tail

• move only in one direction along microtubule

  • Dyneins move toward "-” ends (e.g. toward nucleus)

    -cause cilia to bend by sliding microtubules past each other

  • Kinesins move toward “+” ends (away from nucleus)

Intermediate Filaments

• Structural role

• Stable rope-like filaments (8-10 nm)

  • Unlike most microtubules and actin filaments, they do not typically disappear and re-form (Generally lack dynamic instability)

  • Exception: Lamins in nucleus disassemble before mitosis

• Functions

  • Maintain cell shape

  • Cell-cell junctions

  • Cell-matrix junctions

  • Cell-cell communication

tissue types

The type of intermediate filament varies by ____ ____

• epithelial cells = cytokeratins

• mesenchymal cells (connective tissue, muscle) = vimentin & desmin

• nerve cells = neurofilaments

• nucleus (all cells) = lamins

Brown color = presence of cytokeratin = epithelial tumor → carcinoma

Pathologists can often identify a tumor’s tissue of origin by examining how its cells are arranged, but some tumors are too poorly differentiated to determine this by appearance alone. Knowing the origin is important because it influences treatment choices (e.g., radiation for connective tissue tumors, chemotherapy for epithelial tumors). To help identify the tissue type, immunohistochemistry is used: antibodies tagged with peroxidase bind to specific intermediate filaments in the cells. When a substrate is added, peroxidase produces a brown color wherever binding occurs. For example, if cells stain brown with a cytokeratin antibody, the tumor is confirmed to be epithelial in origin, though further tests are needed for a more specific diagnosis.

Cytoskeleton Functions (Summary)

Stabilizes plasma membrane & maintains cell shape  (A)

Hold cells together (A; I)

Anchor organelles (A; T)

Movement of vesicles (A; T)

Cell movement (A)

Changes during mitosis

–Segregation of chromosomes (T)

–Pinching cell apart into two new cells (A)

******************************************************

A = actin;  I = intermediate filaments;  T = microtubules

Pink lines represent actin filaments;  Green lines represent microtubules

Motor proteins are needed to move vesicles

Inclusions

_____ are inert, NOT metabolically active

Examples

• melanin

• lipofuscin (wear and tear pigment)

• hemosiderin (iron stores; from hemoglobin breakdown)

• glycogen (glucose stores)

• lipid

Melanin

____ produced by melanocytes but transported into adjacent epithelial cellls

• grossly: brown to black (eumelanin) or yellow-reddish (pheomelanin)

eumelanin

brown to black

pheomelanin

yellow-reddish

What are the functions of melanin

• protection against UV radiation, heat, and chemical damage

• coat and feather coloration

• ink used by many cephalopods

In which normal canine tissues might you find melanin pigment?

• hair

• lips

• skin

• eyelids

• gums

Microscopic appearance of melanin depends on stain used. In H&E stain, it is a _____ pigment

brown

Microscopic appearance of melanin depends on stain used. In a Wright (Romanowsky) stain, it is a _____ pigment

often dark green, green-grey

Lipofuscin

“wear & tear pigment”

• From oxidative breakdown of mitochondria and lysosomal digestion

• Common in cells with high metabolic rate

  • Liver

  • Neurons

  • Muscle

• Appearance depends on stain used

  • H&E: Brown pigment

  • Wright (Romanowsky) stain: dark green

****If they look the same, how do you know if it is melanin or lipofuscin (—> based off of location/what cell is the pigment in? = if epithelium, more than likely it is melanin)

Hemosiderin

iron storage molecule, partly derived from breakdown of RBCs

• usually found in macrophages

  • especially in spleen +/- bone marrow

• seen in any tissue after hemorrhage

• appearance depends on stain

  • fixed H&E stain: yellow-brown chunky pigment

  • cytology smear Wright stain: blue-green

  • Prussian blue stain can be used to stain the iron turquoise

glycogen

storage form of glucose

• not stained by H&E (appears as pale areas or vacuoles)

• stains for glycogen

  • Carmin (red) - specific for glycogen

  • PAS (pink) - stains all carbohydrates

Lipid

fat droplets

• found in

  • adipocytes (fat cells)

  • steroid hormone producing cells

  • some types of glands

• extracted during processing so appear vacuoles