Image of a fluorescently-labeled growth cone extending from an axon F-actin (red) microtubules (green).
A growth cone is a large actin-supported extension of a developing or regenerating neurite seeking its synaptic target. Their existence was originally proposed by Spanish histologist Santiago Ramón y Cajal based upon stationary images he observed under the microscope.
He first described the growth cone based on fixed cells as "a
concentration of protoplasm of conical form, endowed with amoeboid
movements" (Cajal, 1890). Growth cones are situated on the tips of neurites, either dendrites or axons, of the nerve cell.
The sensory, motor, integrative, and adaptive functions of growing
axons and dendrites are all contained within this specialized structure.
Structure
Two
fluorescently-labeled growth cones. The growth cone (green) on the left
is an example of a “filopodial” growth cone, while the one on the right
is a “lamellipodial” growth cone. Typically, growth cones have both
structures, but with varying sizes and numbers of each.
The morphology of the growth cone can be easily described by using
the hand as an analogy. The fine extensions of the growth cone are
pointed filopodia known as microspikes. The filopodia are like the "fingers" of the growth cone; they contain bundles of actin
filaments (F-actin) that give them shape and support. Filopodia are the
dominant structures in growth cones, and they appear as narrow
cylindrical extensions which can extend several micrometres beyond the
edge of the growth cone. The filopodia are bound by a membrane which
contains receptors, and cell adhesion molecules that are important for axon growth and guidance.
In between filopodia—much like the webbing of the hands—are the "lamellipodia".
These are flat regions of dense actin meshwork instead of bundled
F-actin as in filopodia. They often appear adjacent to the leading edge
of the growth cone and are positioned between two filopodia, giving
them a "veil-like" appearance. In growth cones, new filopodia usually
emerge from these inter-filopodial veils.
The growth cone is described in terms of three regions: the
peripheral (P) domain, the transitional (T) domain, and the central (C)
domain. The peripheral domain is the thin region surrounding the outer
edge of the growth cone. It is composed primarily of an actin-based cytoskeleton, and contains the lamellipodia and filopodia which are highly dynamic. Microtubules,
however, are known to transiently enter the peripheral region via a
process called dynamic instability. The central domain is located in
the center of the growth cone nearest to the axon. This region is
composed primarily of a microtubule-based cytoskeleton, is generally
thicker, and contains many organelles and vesicles of various sizes. The transitional domain is the region located in the thin band between the central and peripheral domains.
Growth cones are molecularly specialized, with transcriptomes and proteomes that are distinct from those of their parent cell bodies.[3]
There are many cytoskeletal-associated proteins, which perform a
variety of duties within the growth cone, such as anchoring actin and
microtubules to each other, to the membrane, and to other cytoskeletal
components. Some of these components include molecular motors that
generate force within the growth cone and membrane-bound vesicles which
are transported in and out of the growth cone via microtubules. Some
examples of cytoskeletal-associated proteins are Fascin and Filamin (actin bundling), Talin (actin anchoring), myosin (vesicle transport), and mDia (microtubule-actin linking).
Axon branching and outgrowth
The
highly dynamic nature of growth cones allows them to respond to the
surrounding environment by rapidly changing direction and branching in
response to various stimuli. There are three stages of axon outgrowth,
which are termed: protrusion, engorgement, and consolidation. During
protrusion, there is a rapid extension of filopodia and lamellar
extensions along the leading edge of the growth cone. Engorgement
follows when the filopodia move to the lateral edges of the growth cone,
and microtubules invade further into the growth cone, bringing vesicles
and organelles such as mitochondria and endoplasmic reticulum.
Finally, consolidation occurs when the F-actin at the neck of the growth
cone depolymerizes and the filopodia retract. The membrane then
shrinks to form a cylindrical axon shaft around the bundle of
microtubules. One form of axon branching also occurs via the same
process, except that the growth cone “splits” during the engorgement
phase. This results in the bifurcation of the main axon. An additional
form of axon branching is termed collateral (or interstitial)
branching.
Collateral branching, unlike axon bifurcations, involves the formation
of a new branch from the established axon shaft and is independent of
the growth cone at the tip of the growing axon. In this mechanism, the
axon initially generates a filopodium or lamellipodium which following
invasion by axonal microtubules can then develop further into a branch
extending perpendicular from the axon shaft. Established collateral
branches, like the main axon, exhibit a growth cone and develop
independently of the main axon tip.
Overall, axon elongation is the product of a process known as tip
growth. In this process, new material is added at the growth cone
while the remainder of the axonal cytoskeleton remains stationary. This
occurs via two processes: cytoskeletal-based dynamics and mechanical
tension. With cytoskeletal dynamics, microtubules polymerize into the
growth cone and deliver vital components. Mechanical tension occurs
when the membrane is stretched due to force generation by molecular
motors in the growth cone and strong adhesions to the substrate along
the axon. In general, rapidly growing growth cones are small and have a
large degree of stretching, while slow moving or paused growth cones
are very large and have a low degree of stretching.
The growth cones are continually being built up through construction of the actin microfilaments and extension of the plasma membrane via vesicle
fusion. The actin filaments depolymerize and disassemble on the
proximal end to allow free monomers to migrate to the leading edge
(distal end) of the actin filament where it can polymerize and thus
reattach. Actin filaments are also constantly being transported away
from the leading edge by a myosin-motor driven process known as
retrograde F-actin flow. The actin filaments are polymerized in the
peripheral region and then transported backward to the transitional
region, where the filaments are depolymerized; thus freeing the monomers
to repeat the cycle. This is different from actin treadmilling since
the entire protein moves. If the protein were to simply treadmill, the
monomers would depolymerize from one end and polymerize onto the other
while the protein itself does not move.
The growth capacity of the axons lies in the microtubules which
are located just beyond the actin filaments. Microtubules can rapidly
polymerize into and thus “probe” the actin-rich peripheral region of the
growth cone. When this happens, the polymerizing ends of microtubules
come into contact with F-actin adhesion sites, where microtubule
tip-associated proteins act as "ligands". Laminins of the basal membrane interact with the integrins
of the growth cone to promote the forward movement of the growth cone.
Additionally, axon outgrowth is also supported by the stabilization of
the proximal ends of microtubules, which provide the structural support
for the axon.
Axon guidance
Model
of growth cone-mediated axon guidance. From left to right, this model
describes how the cytoskeleton responds and reorganizes to grow towards a
positive stimulus (+) detected by receptors in the growth cone or away
from a negative stimulus (-).
Movement of the axons is controlled by an integration of its sensory
and motor function (described above) which is established through second
messengers such as calcium and cyclic nucleotides. The sensory function
of axons is dependent on cues from the extracellular matrix which can
be either attractive or repulsive, thus helping to guide the axon away
from certain paths and attracting them to their proper target
destinations. Attractive cues inhibit retrograde flow of the actin
filaments and promote their assembly whereas repulsive cues have the
exact opposite effect. Actin stabilizing proteins are also involved and
are essential for continued protrusion of filopodia and lamellipodia in
the presence of attractive cues, while actin destabilizing proteins are
involved in the presence of a repulsive cue.
A similar process is involved with microtubules.
In the presence of an attractive cue on one side of the growth cone,
specific microtubules are targeted on that side by microtubule
stabilizing proteins, resulting in growth cone turning in the direction
of the positive stimulus. With repulsive cues, the opposite is true:
microtubule stabilization is favored on the opposite side of the growth
cone as the negative stimulus resulting in the growth cone turning away
from the repellent. This process coupled with actin-associated
processes result in the overall directed growth of an axon.
Growth cone receptors detect the presence of axon guidance molecules such as Netrin, Slit, Ephrins, and Semaphorins. It has more recently been shown that cell fate determinants such as Wnt or Shh
can also act as guidance cues. The same guidance cue can act as an
attractant or a repellent, depending on context. A prime example of this
is Netrin-1, which signals attraction through the DCC receptor and
repulsion through the Unc-5 receptor. Furthermore, it has been
discovered that these same molecules are involved in guiding vessel
growth. Axon guidance directs the initial wiring of the nervous system
and is also important in axonal regeneration following an injury.
