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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Axonal initial segment

Information flows along a neuron in one direction i.e. synapse to cell body, then to axon initial segment (AIS), from their its passed to Axon and then to terminal region. Actually, neurons will receive both excitatory and inhibitory synaptic inputs on their cell bodies and dendrites. The fire of the repeated synaptic inputs will be summated and bursted out as action potential which will be taken cared by AIS. Axonal initial segment (AIS) is made up of high densities of voltage gated Na⁺ and K⁺ channels which initiate and modulate action potential. In the myelinated axons of vertebrates, the propagation of action potential is rapid along the axon through the activated cluster of sodium channels at the node of Ranvier. The action potential reaches the axon terminal and helps in release of neurotransmitters to propagate the signal across the synaptic cleft to another cell. 

Fig 1: Show the sequence of information follow through the neuron from the synapses. The AIS can be seen soon after the beginning of the axon (green) which consists of node of Ranvier. Node of Ranvier is found at different regions of the axon which consists of cluster of sodium channels.  

Both AIS and node of Ranvier consists of ion channels, cell adhesion molecules, extracellular matrix molecules, proteins like kinases, accessory proteins and cytoskeleton scaffolds.

The axonal initial segment is enriched with sodium gated channels which facilitates a high sodium current density and a low action potential threshold.

The assembly of AIS is actually an intrinsic property of neuron where no extracellular or glial dependent cues are required. But the formation and localization of node of Ranvier is regulated by glial derived signals. 

Ankyrin G

A cytoskeleton scaffold protein which is a master organizer of membrane domains and subcellular polarity in many cell types. This protein is restricted to AIS and AIS targeting motif located in the cytoplasmic loop of the neuron which connects the domain II and III of sodium channels binding to AnkG.  Sodium channel and AnkG interaction is facilitated by Phosphorylation of AIS targeting motif by casein kinase II (CK2), which is enriched at the AIS and node of Ranvier.

Removal or silencing of AnkG protein will lead blocking the clustering of sodium channels at AIS. And also it will lead to blocking the subcellular polarization of potassium channels KCNQ2 and KCNQ3, the cell adhesion molecules neurofascin and neuronal cell adhesion molecules NrCAM, the AIS extracellular matrix and the cytoskeleton protein IV spectrin.

From this we can learn that AnkG functions as a scaffold to which all other AIS proteins are tethered directly or indirectly and consequently AnkG establishes the subcellular polarity of these molecules.

The voltage gated sodium channel Nav1.6 and Nav 1.2 are found in the distal and proximal AIS.  The Kv1 family is also found primarily in the distal AIS.   

Fig 2: shows the structure of AIS 

 Axonal initial segment is composed of the following thing.

1. Ion channels (Nav1.x, KCNQ2–KCNQ3 and Kv1.x)
2. Neuronal cell adhesion molecule (NrCAM)
3. Neurofascin 186 (NF186)
4. Disintegrin and metalloproteinase domain-containing protein 22 (ADAM22)
5. Transient axonal glycoprotein 1 (TAG1, also known as contactin 2)
6. CASPR 2
7. Extracellular matrix molecules (brevican and versican)
8. Cytoskeletal scaffolds (AnkG, "IV spectrin and postsynaptic density protein 93 (PSD93))

Protein with unknown role in AIS

1. Casein kinase II (CK2)
2. Phosphorylated nuclear factor-κB (pNFκB)
3. Phosphorylated inhibitor of κBα (pIκBα)

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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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