Cell Morphology and Motility

Peter Takizawa Cell Biology Cell morphology facilitates cell function.

Our bodies contain over 200 diferent types of cells. Most of those cells have a unique shape, size and structure to facilitate their biological functions. For example, neurons must relay signals between cells and often over long distances. To accomplish this, neurons extend long processes called axons and dendrites that send and receive signals, respectively, from other cells. Dendrites and axons can be hundreds of times the length of the cell body. To maintain these structures, neurons need to provide structural support and deliver biochemical components along the length of axons and dendrites.

Enterocytes are cells that line the inner lining of the small intestine. Their primary function is to absorb nutrients from the lumen of the intestine and release them to the blood stream. To increase the efciency of the uptake of nutrients, enterocytes form many small projections of the cell membrane, called microvilli, into the lumen of the intestine. This increases the overall surface area of the enterocyte. • Actin ﬁlaments

• Myosin Filaments

• Intermediate ﬁlaments

• Cell motility Actin ﬁlaments support and modify cell morphology.

Actin Microtubules

The actin cytoskeleton plays a critical role in cell morphology. It provides structural and mechanical support to plasma membrane, stabilize interactions between cells and between cells and the ECM. It also allows cells to change their morphology and to move. Actin ﬁlaments are a polymer of a single protein.

A reminder of some of the key features of actin and actin filaments: 1.Single globular protein ~ 43 kD. 2. Binds and hydrolyzes ATP. 3. Contains cleft on one side of protein for ATP. 4. Polymerizes into filaments. 5. Filaments are helical. 5.1. 36 nm pitch. 5.2. Lateral interaction increase strength. 5.3. A few micrometers in length -> shorter than MTs and less rigid. Actin filaments crosslinked into networks to increase mechanical strength.

Individual actin filaments are relatively weak and lack rigidity to provide strong mechanical support. Strength is generated by crosslinking actin filaments into networks. Cells can arrange actin filaments into diferent types of networks that serve diferent functions in the cell. A web-like arrangement of filaments provides broad support to the cell membrane. A tightly packed array of filaments allows cells to extend projections of the cell membrane. Actin crosslinking proteins generate different networks of filaments.

Fimbrin α-actinin Filamin

To create diferent arrangements of actin filaments cells employ a variety of actin crosslinking proteins.Actin crosslinking proteins contain two actin binding domains (ABD) or are dimers of a protein with an actin-binding domain. The space between the two actin-binding domains determines arrangement of the filaments. A short space between actin-binding domains generates tightly packed filaments, whereas a longer space between actin-binding domains creates for loosely packed filaments. If the domain between the actin-binding domains is flexible it can generate a web-like arrangement of actin filaments. Web of actin filaments supports the cell membrane.

For example, filamin is a dimer of two proteins each with an actin-binding domain. The region of filamen that forms a dimer is flexible and allows filament to crosslink actin filaments at angles to each other.This creates a web of actin filaments that provides support to a large area of the cell membrane. Microvilli supported by parallel arrays of long actin filaments.

To generate structures like microvilli, cells use fimbrin which has a short gap between its two actin-binding domains. Fimbrin creates a tightly packed, parallel array of actin filaments that to generate dense, parallel arrays of actin filaments that provide increased mechanical strength. Myosins generate changes in cell shape and transport vesicles and organelles.

• Muscle contraction.

• Cell shape and motility.

• Cytokinesis.

• Organelle transport.

Actin filaments not only provide stability for static structures such as microvilli but also allow cells to change shape. Actin filaments can be used by myosin filaments to generate tension on the cell membrane and cause cells to contract. Examples are contraction of all muscle cells, contraction of cells during wound healing, and cytokinesis. Bipolar myosin filaments generate force of contraction.

The myosin that forms filaments, called muscle myosin, is similar to myosin that transport organelles except that it lacks a domain for binding organelles. In addition, muscle myosin has a longer coiled coil domain that allows it to polymerize into filaments. One important feature of myosin filaments is that they are bipolar as the motors one side of the filaments want to move in a direction opposite to the motors on the other end of the filament. Calcium triggers contraction of actin and myosin filaments.

The bipolar arrangement of myosin filaments allows them to pull on two diferent actin filaments. If these actin filaments are attached to diferent regions of the cell membrane, the myosin filament will cause the cell to contract at these two regions. Contraction by myosin filaments is regulated by calcium. When calcium levels are low, the myosin is inactive. When calcium concentration increases, the myosins become active and start pulling on the filaments, causing cell to contract. When calcium levels fall, the myosins lose activity, releasing the actin filaments and relaxing the cell. Calcium triggers contraction of actin and myosin filaments.

Ca2+

Intermediate filaments are the third component of the cytoskeleton but less well studied than actin and microtubules. Intermediate filaments primarily provide mechanical support and don’t support transport of material by motor proteins. Intermediate filaments integrate cells into a mechanical network.

Intermediate filaments extend from the nucleus to the cell membrane. At the cell membrane they Interact with proteins that bind proteins in the cell membranes of neighbor cells. The proteins in the neighbor cells are also linked to intermediate filaments. In this way, intermediate filaments are integrated into large network that spans a group of cells, increasing the mechanical strength of the cells and tissue and protecting it against external stress. For this reason intermediate filaments are found predominantly in cells that face significant mechanical stress, such as skin. Intermediate filaments provide tensile strength.

Kreplak et al. 2008

The unique properties of Intermediate filaments is that they respond diferently to low and high forces. At low forces intermediate filaments will stretch but as the force increases, intermediate filaments become less flexible and start resisting the force. This property makes intermediate filaments more mechanically robust than either microtubules or actin filaments. Intermediate filaments provide tensile strength.

SImilar to microtubules and and actin filaments, intermediate filaments are polymers that from from smaller subunits. However, the structure of the base subunit of intermediate filaments is very diferent from actin or tubulin. First, it is not a globular protein but contain a long helical region or coiled coil. The helical region mediates interaction with another intermediate filament protein. The dimer contains globular domains on either end. Two dimers associate in head to tail fashion with slight ofset. The tetramer is considered the base subunit of intermediate filaments. Note that the base subunit lacks the inherent polarity of actin or alpha-beta tubulin. Consequently, intermediate filaments are not polarized as are actin filaments and microtubules. Soluble base subunit of IFs.

Tetramers polymerize end on end to form protofilaments. 8 protofilaments twist together to form an intermediate filament. The extensive lateral interactions are in part what gives intermediate filaments their tremendous strength. Intermediate filaments assemble through coiled coil interactions.

NH2 COOH

COOH NH2

Keratin Cytokeratin 19 Vimentin

Neuroﬁlaments

Lamin A Lamin B

Intermediate filaments comprise large family of proteins. Keratin is the largest class with ~50 members and found prominently in skin and hair. Neurofilaments are found in axons and lamins localize to the inner nuclear membrane. Mutations in intermediate filaments cause blistering diseases.

The importance of intermediate ﬁlaments to the mechanical stability of cells and tissues is illustrated by mutations in keratin genes. Keratins are the intermediate ﬁlaments found in skin cells and are critical maintaining the structural integrity of the skin. Mutations in keratin genes weaken the interactions between neighbor cells. Consequently, mild abrasion to skin cause the layers of the skin to separate leading to blisters. One cause of the skin-blistering disease epidermolysis bullosa is mutations in keratin genes. Cell Motility

Some cells can move by crawling along a surface. Neutrophils chase bacteria through tissues or across a substratum.

Neutrophils track and chase a bacterium. Neutrophils move at ~ 0.1 to 0.2 µm/s. Metastatic cancer cells crawl through tissues and along a substratum.

Metastatic cells use the same crawling ability to escape from a localized cancer and enter the lymphatic or circulatory system and spread to other organs. Cells can follow the concentration gradient of a single chemical.

When cells move, they usual follow some external chemical that guides the direction of their movement. Cell motility involves three steps: pushing, attaching, pulling.

Cell motility involves several steps. To move across a surface, cells generate force that pushes forward a broad section of the plasma membrane. This is called the leading edge and extends a part of the cell in certain direction. Attachments between the cell and the substratum give the cell something to push against and stabilizes the leading edge. The cell pulls on attachments at back end of cell to detach them from the substratum. This allows the cell to move its backend forward toward the leading edge. Polymerization of short, branched ﬁlaments pushes forward the plasma membrane.

Pushing of the cell membrane is generated by polymerization of actin filaments. Motile cells have a high concentration of actin at their leading edge. Higher resolution images of the leading edge reveal a dense network of actin filaments that are short and branched. Polymerization of short, branched filaments pushes forward the plasma membrane.

Pushing of the cell membrane is generated by polymerization of actin filaments. Motile cells have a high concentration of actin at their leading edge. Higher resolution images of the leading edge reveal a dense network of actin filaments that are short and branched. Polymerization of new actin filaments, but not from existing filaments, has a lag phase.

+ preformed ﬁlaments

Time Time

To initiate motility, cells must regulate when and where they polymerize actin. They do this use actin nucleating factors. Normally, there is a delay in the polymerization of actin called the lag phase. However, in the presence of preformed ﬁlaments or nucleating factors, there is no delay in actin polymerization. By controlling the activity and location of nucleating factors, the cell can specify where actin polymerization occurs in the cell. ARP2/3 resembles actin dimers and nucleates ﬁlament formation to overcome lag phase.

For cell motility, the most important nucleating factor is the ARP2/3 complex. The ARP2/3 complex is set of proteins, two of which resemble actin. When one actin monomer associates with ARP2/3, it forms a stable platform for ﬁlament growth. Actin ﬁlaments grow from their plus ends.

Like microtubules, actin filaments are polarized. The plus end of actin filaments is the fast growing end as monomers are readily incorporated into this end. Minus ends add monomer very slowly and often shrink. Minus ends are often stabilized by nucleating factors. Capping proteins control the length of actin filaments. Polymer concentration

Actin concentration

Cell motility depends upon forming short filaments because long filaments are less rigid and wouldn’t support the cell membrane. Cells control length of actin filaments with capping proteins. Capping proteins bind plus ends and prevent further addition of monomer to stop growth. In the presence of high concentration of capping protein, cells form many short actin filaments. Cofilin severs actin filaments leading to depolymerization.

To support the continuous pushing forward of the cell membrane, the cell needs a continuous supply of actin monomers to polymerize new filaments. To maintain a supply of actin monomer, the cell recycles old filaments that are no longer required to support cell motility. Cells recycle old filaments by severing them and allowing them to depolymerize. Cofilin binds to the sides of actin filaments and induces a twist in the filament. The twist causes the filament to sever, exposing a minus end from which the filament depolymerizes.

To diferentiate old from new filaments, cofilin only binds to filaments with actin that is bound to ADP. When actin is incorporated into filaments, it is bound to ATP. Thus, new, growing filaments will contain mostly actin-ATP. Over time, actin hydrolyzes ATP to ADP. When most of the filament is actin-ADP, cofilin can bind and sever the filament. Polymerization of short, branched filaments pushes forward the plasma membrane.

Pushing of plasma membrane generated by polymerization of actin filaments. High concentration of actin at leading edge. Higher resolution reveals dense network of filaments. Short. Branched. Arp2/3 and capping protein generate short, branched filaments.

Coﬁlin

Cells use a combination of ARP2/3 and capping protein to generate short, branched filaments. ARP2/3 nucleates the polymerization of new filaments by binding to the side of existing filaments. The new filaments grow at ~ 70˚ angle to the existing filament. This creates a network of branched filaments. The high concentration of capping protein ensures that the filaments remain short. The continuous activation of ARP2/3 generates an expanding network of branched filaments that pushes forward the cell membrane. Toward the center of the cell cofilin severs old filaments to generate a constant supply of actin monomer for new filament growth. External signals control location of actin polymerization via activation of ARP2/3.

WASp

Cells move in the direction of an external signal and must be able to integrate the location of the external signal with the proteins that regulate actin polymerization. A central component in this linkage is WASp (Wiskott-Aldrich Syndrome protein). WASp is activated by receptors in the cell membrane that bind external signaling molecules. Active WASp associates with ARP2/3 and activates it, triggering the polymerization of actin ﬁlaments and cell motility. Bacterial molecules activate signaling pathway in neutrophils to initiate actin polymerization.

ARP2/3

Neutrophils contain receptors that bind to bacterial peptides (peptides with formyl groups). When these receptors bind bacterial peptides, they activate ARP2/3 triggering the growth of a branched network of actin ﬁlaments. This pushes the cell membrane towards the source of the bacterial peptides. Take home points...

• Actin and myosin ﬁlaments allow cell to form diﬀerent morphologies

• Intermediate ﬁlaments provide robust mechanical resistance

• Coordinated actin polymerization pushes forward the cell membrane to drive cell motility