What mechanisms drive cytoskeleton extension within podosomes? 2018-02-06T10:40:35+00:00

What mechanisms drive cytoskeleton extension within podosomes?

Interactions between the extracellular matrix (ECM) and cell surface integrins leads to podosome formation. The initiating signal is transduced through mechanosensing integrins to the cytoskeleton, upon which the actin network undergoes significant re-organization to promote formation of the podosome. This process begins with actin nucleation.

It is widely believed that Arp2/3-mediated nucleation is the major means by which the podosome actin cytoskeleton is built. This is evidenced by the colocalization of actin, Arp2/3 and the potent activator of Arp2/3, WASP (Wiskott Aldrich Syndrome protein), to these structures [1], as well reduced podosome formation following sequestration [2] or small molecule inhibition [3] of Arp2/3. Notably, WASP has not been detected in focal adhesions (FAs), highlighting a key point of divergence between the protein constituents of podosomes and FAs [4].

The activity of WASP is central to podosome formation, as illustrated in cells lacking full length WASP that consequently also lack podosomes. Resulting defects include reduced bone resorption in osteoclasts [5] and impaired migration of dendritic cells [6] and macrophages [7]. The migratory defects observed in WASP-deficient immune cells are in large part responsible for the systemic, immunological deficits observed in boys presenting with the X-linked disease, Wiskott Aldrich Syndrome (WAS) [6].

The upstream regulation of WASP-activated, Arp2/3-mediated actin nucleation involves the RhoGTPase, Cdc42. The importance of Cdc42 in podosome formation is evidenced by the mutant protein studies showing constitutively inactive Cdc42 greatly reduces podosome formation [6]. The mechanism underlying this is reliant on the interaction between Cdc42 and WASP, which activates the Arp2/3 complex [8]. It should be noted that Cdc42 is suggested to have a dual role in podosome formation [6], firstly in promoting actin nucleation and secondly in determining the distribution of podosomes in migrating cells. The latter function is evidenced by the expression of constitutively active Cdc42 resulting in defects in podosome clustering and their polarized arrangement along the length of the cell [6]. Cdc42 activity must therefore be tightly regulated in order to promote both of these activities.

In addition to the known activation of Arp2/3 by Cdc42-WASP, a second weaker activator of Arp2/3 also resides in podosomes (and invadopodia), namely cortactin. Although cortactin is key to the formation of invadopodia, with depletion of this protein greatly diminishing the number of invadopodia [9] its importance in podosome formation is less clear. In leukocytes, a primary model for the study of podosomes, the cortactin homologue HS-1 (hematopoietic lineage cell-specific protein 1) is dispensable for podosome formation but is instead required for the polarized distribution of podosomes during migration [10]. Cortactin is also suggested to be required to transport vesicles for matrix metalloproteinase-mediated degradation of the ECM. This is dependent on the cortactin-binding domain of WIP (WASP interacting protein), a protein essential for WASP-mediated actin polymerization during podosome formation [11].

Collectively these studies support Arp2/3-mediated nucleation as the primary means for initiating construction of the podosome architecture. Electron microscopy (EM) studies have detected branched actin filaments comprising both the actin core [12] and the radial actin network [13] – though in the latter instance there is still no clear consensus on whether the filaments are indeed branched or not [12].

There is some speculation over the involvement of formin-mediated nucleation of actin within podosomes although the investigations into this are still in their infancy. Evidence for formin-mediated nucleation in podosomes comes from a single study that has observed the formin, FRL1, localizing to a cap-like structure on top of the actin core of macrophage podosomes [14]. Further investigation is needed to confirm this finding and resolve the issue of its apparent localization to the pointed ends of the actin filaments, as opposed to the barbed ends where formins are conventionally known to act. It should be noted that a model for the polarity of radial actin filaments of podosomes has been posited, whereby the barbed ends are orientated towards to the actin core of the podosome, as inferred from scanning EM in conjunction with myosin ‘S1’ fragment labeling [12].

In this model actin polymerization, at the face of the actin core, generates forces that push against the core and consequently promotes podosome protrusion [12]. In this context, it is plausible that formins, localizing as expected at the barbed ends of actin filaments, could be found encasing the tops of the actin cores. However it is important to note that this model is yet to be comprehensively tested and therefore remains a speculative hypothesis. The prevailing theory for actin assembly during podosome formation therefore still centers around Arp2/3-mediated nucleation.

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