Microtubules: Role in axon and dendrites formation

Polarized cells such as neurons have two distinct domains i.e. molecular and functional domains which have been emerged from the cell body. In neurons, axon and dendrites are two distinct functional units emerged from the cell body by the process called neuronal polarity. Axons are thin, single and long structure which will transmit the signals to the target or synaptic partner. Whereas dendrites are shorter, multiple structures which has the ability to receive signals from different regions of cells or axons.

Before getting with role of microtubules in axon and dendrite formation, we need to look into the neuronal polarity part which is already been explained in one of my post. I.e. a hippocampal neuron polarize in the presence of insulin like growth factor (IGF-I) without any cell- cell interaction or extracellular matrix. Now these polarized cells exhibit the molecular features that distinguish axon and dendrites.

So in neuronal polarity, these are the list of protein involved:

1) Kinases

2) Phosphatase  

3) Small GTPase

4) Scaffolding proteins

Now what microtubule has to do with neuronal polarity?

Actually if polarity is lost, then it could be due to some modification in microtubule organization and dynamics. Hence microtubule has a very important role in neuronal polarity.

When do we say that neuronal polarization takes place?

It happens to place when there is local microtubule assembly and stabilization in one of the neurite. Change in the dynamics of the microtubule will lead to alteration in axon and dendrite specification.

Facts about microtubules:

Hollow cylindrical, covalent cytoskeleton polymers (24 nm diameter)

Made of tubulin proteins (α tubulin and β tubulin)

For polymerization Tubulin dimers bound to GTP

For depolymerization Tubulin dimers bound to GDP

α tubulin and β tubulin forms dimer

γ tubulin are attached at the minus end by nucleation. They form a ring at the minus which act as a cap to prevent further addition of any tubulin at the site.

    Microtubule structure

Two ends of microtubules: plus end (+) and minus end (-)

At Plus end (+), dimers with GTP are assembled and GTP bound to β tubulin gets hydrolyzed to GDP through inter dimer contacts. Growth and shrinkage of microtubule takes at this end.

At minus end (-), dimers gets dissociated and polymerization at this end is prevented by cap formed by γ-tubulin ring complex. Basically, minus ends are anchored to the centrosome or may be found as free ends in some cells.

Function: cell motility, mitosis, intracellular transport, secretion, maintenance of cell shape and cell polarization. 

Cycle of microtubule growth and shrinkage

Microtubule has the ability to undergo the cycle of growth and disassembly (as shown in the fig). They never reach the steady state length, but they exist in either polymerization or depolymerization state.  

There are two things happen in this cycle. One is catastrophe where depolymerization of the tubulin dimers takes. And other is rescue where the dimers polymerize together for the growth. 

These two things depend on the binding of GTP at the E site (nucleotide exchangeable site) of the β tubulin. During polymerization, the E site of the β tubulin has to be bound with GTP so that α tubulin can come and bind to it. Polymerization dynamics of microtubules are important for their biological functions because they allow themselves to rapidly reorganize, differentiate spatially, temporally in accordance with cell context, and to generate pushing and pulling forces during polymerization and depolymerization. 

Microtubule Associated proteins (MAPs)

These are the protein which joins the two microtubules together and form a bundle. There are two MAPs which are present in the axon and dendrites:

a) Tau protein

b) MAP2

a) Tau protein

Microtubules in axon has uniform orientation with their plus ends facing the axon tip and it’s bundled up together by the protein called tau protein. It’s also present in the somato-dendritic compartment and axon region. It’s not having any essential role in axon growth and microtubule stability. This statement was actually studied in tau knock out mice where its neuron showed no significant difference with that of the wild type mice. Except in one case where the tau deficient mice showed reduction in packing density and number of microtubules in cerebellar parallel fibers, a small type of caliber axon.

But this protein is important in the case of neurite outgrowth as well as growth cone motility. Studies have been taken place in chick sensory neuron (dorsal root ganglion) where inactivation of tau protein leads to reduction in the number and length of neurite.  In the growth cone, inactivation of tau protein lead to 20% decrease in the lamellopodial size and there lamellopodial motility is affected.     

Tau protein plays a vital role in development of Alzheimer’s disease.  

b) MAP2

Microtubules in dendrites have multiple orientations with their plus ends facing either the cell body or the dendritic tips and the bundling protein here is the MAP2.  It has 3 or 4 microtubule binding domains at the COOH- terminal, and is involved in microtubule assembly and stabilization in dendrites. MAP2 is an anchoring protein of PKA in dendrites, whose loss leads to reduced amount of dendritic and total PKA and reduced activation of CREB.

Fig 3: Developed Neuron: Microtubules in Axon and dendrites are shown by magnifying the parts. Tau in axonal microtubule and MAP2 in somatic dendritic compartment. organelles such golgi apparatus,  polyribosomes and Endoplasmic reticulum are localized in the dendritic region.(Nature Neuroscience review)

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Growth cone and its role in axonal guidance

Growth cone are specialized and highly motile cellular compartment at the tips of the growing axon which supports the growth of the axon by sensing the extracellular cues and transducing it to the cytoskeletons.

The structure consists of a central region filled with organelles and microtubules; whereas at the peripheral region, it has highly dynamic, actin rich region such as Lamellipodia and Filopodia.

Lamellipodia are the broad veil like cellular protrusions that contain branched actin filaments. Filopodia are thin protrusions made out of unbranched and parallel F actin bundles. 

Fig 1: Shows that the central region of the structure is composed of densely concentrated organelles, whereas thin veil like Lamellipodia and spiky Filopodia is found at the peripheral region.  

This is large paused growth cone imaged through differential interference contrast microscope. 

A brief about growth cone and its structure is explained in the topic called Cytoskeleton Role in Neuronal polarity.

A growth cone helps the developing neuron to reach the particular target site or synaptic partner. Hence axonal growth cones used to navigate along the specific pathways in the response to the molecule guidance cues. In this article, it will be explained that how growth cone is influenced by extracellular cues inorder to select a particular pathway towards the target site.

The growth cone is highly concentrated with actin filaments rather than microtubule. Microtubules which are bundled together in the axon shaft will also extend into the central region of the growth cones, but the growth cone stalls it, thereby making it to form a prominent loop in the central region.

Fig 2: distribution of actin and microtubule in the growth cone. 

In the Filopodia region, the actin filaments will constantly undergo polymerization and depolymerization. At the barbed end there will be addition of G actin molecule, whereas the in the pointed end the G actin molecules are constantly removed, thereby leading to the movement and extension of Filopodia. The G-actin molecule is brought back from the peripheral region to the distal region by myosin (actin cargo carrying protein) and this process we term as retrograde movement. The tip of the Filopodia contains the receptors for the extracellular cues and in the Lamellipodia is concentrated with regulators of actin and microtubules which gets activated and deactivated based the signals obtained at the peripheral region of the growth cones. Combining these two properties, growth cone is said to be the compartment which has the ability to guide the neuron to reach its target site or synaptic partner. 

Fig 3: Pictorial representation of growth cone action towards the gradient. 

In the fig 3, it’s shown how the cytoskeletons modify which lead the growth cone to turn towards the highly concentrated gradient. The polymerization and depolymerization of the actin takes place simultaneously in different regions. For eg: In the stage 3, the filaments are stable and polymerize towards the attracting gradient region, whereas there happens to be depolymerization in the non gradient region of the Growth cone. 

Cytoskeletons	Stage 1	Stage 2	Stage 3	Stage 4
Actin	Balanced F actin dynamics across the GC	Increase in anticapping and severing, decrease in capping leading to polymerization	Cross linking of the filament leads to stable ribs.	Capping of the F actin filament will not allow the polymerization and finalize to stabilization.
Microtubules	Random MT exploration of P region	Polymerization happens with less catastrophe	Acetylation and detyrosination leads to stabilization of microtubule.	Bundling

  Table 1: Action of cytoskeletons in the growth cone under two gradient regions. 

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Centrosome role in neuronal polarity

Centrosome or MTOC (Microtubule organizing center) is a kind of organelle found in both plant and animal cell which guides the formation of microtubule in the cell. Microtubule array arises from the Centrosome and spreads all over the structure of the normal cell. But in the case of developing neuron, the Centrosome is localized towards one particular neurite and then the microtubules are guided to that neurite which further forms the axon. Centrosome positioning is an important phenomenon in the neuronal polarity because out of all the neurite; one specific axon has to be formed towards the target cell or neuron.

Centrosome positioning takes place at the stage 3 of neuronal polarity, where the Centrosome is found closer to one particular neurite which is said to be the future axon.

After the cell exits from the cell cycle, it forms neurites. And then certain extracellular cues activate the intracellular cues which take the responsibility of Centrosome positioning. Centrosome positioning is the beginning stage of axon specification. 

Structure of Centrosome is made up of two barrel shaped centrioles and a cloud of pericentriolar material that surrounds them. Microtubules are nucleated from the pericentriolar material and form a radial array emanating away from the Centrosome. Nucleation from the Centrosome regulates key features of the microtubules within the array. First, all of the microtubules assemble with their plus ends away from the Centrosome, resulting in a microtubule array of uniform polarity orientation.

This fig shows that the difference between the nonneuronal and neuronal cell’s centrosome activity. The pluripotent precursor cell can give raise to either nonneuronal cell or neuronal cell. In the nonneuronal cell, a portion of the microtubules nucleated by the centrosome are captured by the leading edge of the cell. The motility of the leading edge pulls on the microtubules, and the attached centrosome reacts by relocating in the direction of cell movement. In the neuron, the microtubules are released, and the centrosome is not relocated. Nevertheless, the microtubules are translocated toward the leading edge, which coalesces into a growth cone. The cell body remains stationary and the microtubules translocate into the space between the cell body and the growth cone, which develops into the axon.  

Microtubules destined for the axon are initiated at the centrosome and then released for translocation. Released microtubules are transported through the cytoplasm with their plus ends leading, and many of these are transported into the axon. In the schematic, the white portions of the microtubules represent the part assembled from the centrosome, while the black portions represent the part assembled after release from the centrosome. Plus ends of microtubules are directed away from the centrosome and toward the distal tip of the axon. The space between the slanted lines through the axon represents hundreds of micrometers of axon growth. During transit, the microtubules elongate specifically from their plus ends.

Still continues.......

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Negative regulators of Neuronal polarity

Negative regulators of neuronal polarity means the molecules will inhibit other downstream or upstream molecules which are involved in controlling the Cytoskeleton formation, stability, endocytosis etc.

PTEN (Phosphatase and tensin homolog)

It is a lipid phosphatase protein which counteracts the enzyme PI3 K (PI3 kinase) by converting PIP3 formed by PI3K into PIP2. Overexpression of this molecule will lead to prevention of PAR 3 localization to neurites and inhibits axon formation. If the PTEN expression is downregulated using SiRNA, then all the neurites becomes axons and there will reduction in dendrite formation. From this we can conclude that PTEN is important for the growth specific single axon. PAR 3 (Drosophila epithelia and neuroblasts) actually directly interacts with PTEN and this property leads to negative feedback loop. Where the PI3Kinase will recruit PAR 3 which in turn brings the PTEN molecule, thus leading to maintenance of PIP3 concentration in axon or PIP2 formed will bind to actin associated proteins such as WASP and regulate the actin formation.

PTEN is associated with PI3K and GSK3β; GSK3β inhibits PTEN by Phosphorylating at its C terminus. Until GSK3β is active, the PTEN activity is downregulated and therefore upregulation of PTEN happens by GSK3β inhibition which might provide a negative feedback regulation by again counteracting at PI3K activity.

Rho A (Rho GTPase Family)

Rho A is a Rho GTPase family protein which has the negative regulation role in the axon stability. Rho A negatively regulates the neurite outgrowth and modulating its signaling pathway will lead to axon regeneration in the central nervous system during injury.  ROCK which is a Rho kinase effector accompanies Rho A in regulating axon growth and actin dynamics during neuritogenesis of cultured cerebellar granule neurons. Rho A-ROCK pathway with profilin II regulates actin and microtubule stability. Still Rho A knock mice is not created inorder to analysis the function of Rho A in neuronal polarity.

GSK3β (Glycogen synthase kinase 3β)

GSK3β is negative regulator of CRMP -2 (collapsin regulated mediator protein-2) which is GSK3β substrate responsible for microtubule polymerization. GSK3β is regulated by Akt protein which is activated by the PIP3 produced by PI3 Kinase. The protein not involved in establishment of neuronal polarity, but also plays a vital role in maintenance of neuronal polarity. CRMP-2 will be phosphorylated at thr-514 by GSK3β thereby making it unable to bind with tubulin dimer and NUMB. Overexpression of active GSK3β will lead to prevention of axon formation, whereas in inactive form it will lead to multiple axon formation. It also inactivates the MAP (microtubule associated proteins) protein by Phosphorylating MAP1B and APC (adenomatous polyposis coil).

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Actin and Microtubules regulators

Actin regulating proteins

Basically the actin dynamics are regulated by actin nucleating, severing, branching and bundling proteins.

Actin nucleating proteins are Arp 2/3 complex (Lamellipodia) and formins (Filopodia). The Arp 2/3 complex is also very important in the formation of Filopodia as well as dispensable for actin organization in neuronal growth cones. 

Growth cone dynamics regulators (directly modulate actin dynamics):

• WAVE (wiskott Aldrich syndrome protein [WASP] family verprolin homologous protein)
• Ena/VASP (Vasodilator stimulated phosphoprotein)
• Profilin
• ADF (actin depolymerization factor)/ cofilin

WAVE protein is localized in the Lamellipodia and is part of the WAVE complex of the proteins that act together to regulate actin polymerization in Lamellipodia. Basically the WAVE pathway regulates the actin polymerization through the Arp 2/3 or Profilin and promotes axon growth. 

A component of WAVE complex called Nap 1 (Nck associated protein 1) which is important in cortical neuronal differentiation as well as proper extension of axon.

The Ena/VASP proteins are localized at the tips of the Lamellipodia and Filopodia, which will accelerate the actin polymerization by their anticapping activity and bundle actin filaments. Absence of these proteins will lead to aberrant actin bundling and failure of Filopodia formation. Therefore formation of neurites in the absences of these proteins is not possible, hence it’s considered important in the neuron formation.

The role of Ena/ VASP signaling in axon formation appears to be conserved throughout the species. Additionally, Ena/VASP proteins recruit the actin regulator profilin, but the physiological relevance of this interaction is still not clear.

Profilin localizes to the leading edges of the growth cones and enhances the formatting of ATP bound G actin monomers that are incorporated into actin barbed ends. Ablation of isoform Profilin IIa protein leads to destabilization of actin cytoskeleton as well as increased in the number and length of the neurites.

But it was investigated in the Profilin Knockout mice, that polarization has taken place normally. It shows that Profilin I is compensating the function of Profilin II, But Profilin I knockout mice are embryonical lethal. 

Cofilin is an actin severing proteins which are also implicated in the neuronal polarity. ADF and cofilin І which are expressed in brain is found to be abundant in the neuronal growth cones. They bind mostly to the ADP-actin rather than ATP actin which results in their association with the pointed end of actin filaments and promoting actin depolymerization.  Both the proteins are regulated by Phosphorylation and phosphorylated form is mostly abundant in the cellular forms. The cofilin proteins are active during axonal growth cones compared to non growing future dendritic growth cones.

Microtubule regulating proteins

CLIP (Cytoplasmic linker proteins) and CLASPs (CLIP associated proteins) regulated the microtubule dynamics.  They act as MT growth promoting and also MT stabilizing protein.

CRMP 2 (collapsin response mediator protein 2) binds to the free tubulin and promotes their capacity to bind to the microtubules. CRMP2 is inhibited by GSK 3β Phosphorylation and PTEN. WAVE complex helps the transportation CRMP2 on kinesin molecule.

Stathmin/Op18 is the MT destabilizing proteins, which can be inactivated inorder to attain MT stability and is necessary for the specification of the nascent axon.  

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