What are podosomes?[Edit]

Podosomes are actin-rich, adhesive structures that are present at the ventral surface of cells of the monocytic myeloid lineage, stimulated endothelial cells [1] and cultured Src-transformed cancer cells. These structures are not limited to the cell periphery, but do exhibit a polarized distribution pattern in migrating cells, localizing to the front at the border between the lamellipodium and the lamellum [2]. Podosome assembly can be described under a series of distinct steps.

Figure 1. Podosomes: (A) Human acute monocytic leukemia cells (THP1) cultured on fibronectin, differentiated towards macrophages with TGF beta1, displaying podosomes (red puncta). Actin was visualized using phalloidin-TRITC. The image is a projection of several Z-planes where lamellipodial actin ruffles on upper planes are false-colored green and actin cores of podosomes on lower planes are inred. (B) Human acute monocytic leukemia cells (THP1) cultured on fibronectin, differentiated towards macrophages with TGF beta1, displaying podosomes (yellow puncta). Actin (red) was stained usingphalloidin-TRITC and vinculin (green) using Alexafluor488-coupled anti-vinculin antibody. The actin core and the surrounding ring complex (vinculin) are clearly visible. The cells were imaged using a DeltaVision microscope. [Images captured by Tee Yee Han, Mechanobiology Institute, Singapore]
Podosomes are adhesive structures and so unsurprisingly they contain many of the same proteins found in focal adhesions (FAs), such as talin,vinculin, paxillin and Src family proteins [3]. An exception to this generality is the protein Tks5 (tyrosine kinase substrate with five SH3 domains), which is found in podosomes and invadopodia, but not in FAs or any other actin-based structures. WASP (Wiskott Aldrich Syndrome protein) shows even greater specificity, being unique to podosomes. Tks5 and WASP can therefore be used as a markers alongside actin, cortactin and the Arp2/3 complex to identify these unique protrusions [4].

Podosome structure[Edit]

Structurally, the podosome is characterized by two main features – an actin core and a ring complex. The actin core contains several coordinators of actin nucleation, namely the Arp2/3 complex and WASP proximal to the plasma membrane and cortactin or HS-1 (hematopoietic lineage cell-specific protein 1) more distally [5]. Further to the dense actin network forming the core, actin filaments emanate radially from the actin core to the plasma membrane and between individual podosomes [6]. 

Figure 2. Podosome structure: (A) The actin core of the podosome (light blue) includes several proteins that promote actin nucleation. The Arp2/3-Cdc42-WASP-WIP complex facilitates actin nucleation and branching proximal to the plasma membrane, whilst cortactin (or its leukocyte-specific homologue HS-1[7]) acts to stabilize branch points more distally [5]. It is not yet clear whether the attachment of the actin core to CD44 receptors occurs via branched or unbranched actin filaments or what protein complex could facilitate this link. Radial actin filaments (dark blue) emanate from the actin core [8] and link to cell surface integrins that form the basis of the adhesive ring complex. The radial network is speculated to exhibit mechanosensory potential [9], involving the transmission of forces in a myosin II dependent manner. (B) The ring complex is formed by a circular array of integrins, to which several other proteins also localize. These proteins include the signaling proteins, Src kinase and Pyk2, the adaptor protein p130CAS, the actin severing protein gelsolin and the focal adhesion-associated proteins talin, vinculin, paxillin and α-actinin. It should be noted that though gelsolin localizes to this ring structure, it is not absolutely required for podosome formation [8].
The ring complex comprises integrins and integrin-associated proteins, such as paxillin [10] and serves to connect cell surface integrins with the cytoskeleton. Originally thought to be a round structure, recent advances in bioimaging have shown the ring complex to have a polygonal shape [11]. These findings were obtained by applying novel super-resolution analysis (Bayesian Blinking and Bleaching (3B) analysis) to data acquired by standard widefield microscopy of cells expressing fluorescently tagged proteins that localize to the podosome ring complex. The increased resolution of the images also suggested that different proteins within the ring complex have distinct localizations, with vinculin appearing more peripherally to talin [11].

In general, the podosome structure is no greater than approximately 0.5 μm in width and 1 μm in depth [10]. The lifetime of this structure is far shorter than that of focal adhesions, lasting only a few minutes [12].

Podosome function[Edit]

Both focal adhesions and podosomes are intimately involved in cell motility, with podosomes specifically implicated in cell invasion. Invasiveness is achieved through the secretion of matrix metalloproteinases (MMPs) from the core of podosomes, which degrade the extracellular matrix (ECM). This promotes motile behaviors that aid a range of processes including; transendothelial migration (diapedesis) of dendritic cells [13], migration of aortic endothelial cells for arterial vessel remodeling [1] and tissue infiltration by macrophages [14]. 

Further evidence for the role of podosomes in cell migration, comes from the immune cells of Wiskott-Aldrich Syndrome patients. These patients lack full length WASP(Wiskott-Aldrich Syndrome protein), which is known to localize to podosomes and is required for their formation. Both the dendritic cells [15] and macrophages [16] of these patients lack podosomes and as a consequence show migratory defects [15, 16]. There is currently speculation that podosomes may also be important in the migration of neural crest cells, due to the neural crest-associated defects seen in Frank-ter Haar patients, who are mutant for the podosome- and invadopodia-specific protein Tks5 [4].

In addition to the roles ascribed above, the idea of podosomes having mechanosensory potential has also been posited [9, 17, 18, 19]. The initiation of podosome formation has been shown to be dependent on the underlying matrix, both in terms of its nature (which ligands are present) and its geometry (whether the ligands are uniformly distributed or arranged in subcellular-sized islands). Cells use different integrin receptors to detect the mechanical constraints of their environment and to decide accordingly whether a podosome should be initiated or not [17]. Following initiation, substrate stiffness continues to play a role in the lifespan of the podosome, with increased stiffness resulting in increased longevity and decreased distance between individual podosomes [18].

Once formed, the podosome itself is hypothesized to exhibit mechanosensory characteristics, transmitting mechanical forces both from the inside-out and outside-in [9]. This was suggested following a series of experiments examining the actomyosin network of the podosome. Myosin II was detected in and around the adhesive ring of the podosome. Changes in the size and shape of the adhesive ring were shown to result in changes in tractional forces against the underlying substrate (inside-out force transmission), in a myosin-dependent manner. Moreover, increasing substrate stiffness, increased the strength of these tractional forces (outside-in force transmission).

Podosomes in osteoclast function[Edit]

Osteoclasts are multinucleated, bone-resorbing cells that use podosomes for bone remodeling. During the process of differentiation from osteoclast precursors to mature osteoclasts, clusters of podosomes rearrange themselves into higher order rings structures, which are finally reorganized into a single belt around the cell periphery [12]. These structures are highly interconnected due to a dense network of radial actin filaments that connect the composite podosomes to each other [20]. 

The structural changes that occur during osteoclast maturation involve the accumulation of F-actin, vinculin, paxillin and α-actinin specifically within podosomes of the forming ring structure [21]. Signaling changes also occur within these podosomes, such as reduced levels of Src-mediated phosphorylation [21]. The podosome protein, cortactin, specifically shows reduced levels of tyrosine phosphorylation, which is suggested to enhance its actin-nucleating activity [22].

Upon bone resorption, the podosome belt is dismantled, leaving behind a ring-like F-actin mesh encompassing the 'sealing zone' [23]. The sealing zone forms the attachment of the osteoclast to the underlying bone and is essential to the process of bone resorption. The inhibition of bone resorption by drug treatment results in loss of the characteristic podosome belt around the cell periphery. Podosomes are therefore believed to play an important role in formation of the sealing zone and bone resorption[23].


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Updated on: Thu, 03 Apr 2014 08:04:08 GMT