V. DISCUSSION
2. LSP1 LOCALIZES TO PODOSOME CAP AND REGULATES MACROPHAGE MIGRATION AND PODOSOME
migra-tion and podosome mechanosensing.
Based on the podosome proteomic analysis described, we selected several pro-teins to screen as potential new components and, among them, decided to focus our attention on the most promising one according to the mass spectrometry score: lymphocyte-specific protein 1 (LSP1).
LSP1 is a protein isolated 30 years ago for the first time in B and T lymphocytes 103. Since then, it has also been found in macrophages, neutrophils, dendritic cells and endothelial cells 87, 88, 104, 105.
In neutrophils and B lymphocytes, it localizes on the cytoplasmic face of the plasma membrane 106, whereas in endothelial cells, it is mostly present in the nu-cleus and decorates F-actin rich microfilaments in the cytoplasm 87, 107. During our studies, it has also been shown, by another group, to co-localize with podosome F-actin cores in dendritic cells, where it also presumably interacts with ARP 2/3 complex and WASP 88.
Previous studies have described LSP1 as a crucial regulator of immune cells chemotaxis, recruitment to inflammation sites and phagocytosis 86, 88, 89, 108-111. For instance, LSP1 overexpression is responsible for neutrophil actin dysfunction (NAD 47/89), an inherited disease where neutrophils have impaired ability to kill bacteria, despite an abundance of hair-like F-actin protrusions, and show reduced adhesion and motility, resulting in severe recurrent infections 112, 113. In contrast, LSP1 deficiency leads to enhanced T cell migration and contributes to the devel-opment of rheumatoid arthritis 90.
LSP1 comprises 339 aminoacids and the structure can be ideally split in two subdomains with a similar number of residues: 1) the N-terminal half, highly acidic in composition, poorly conserved among species and putatively binding Ca2+; 2) the C-terminal half, highly basic in composition, highly conserved among species and harbouring four F-actin binding sites, of which two are caldesmon-like do-mains (CI, CII; weak binding) and two are villin headpiece-like dodo-mains (VI, VII;
strong binding). Interestingly, each isolated domain has the ability to bind F-actin in vitro, however only the cooperation between the two pairs (i.e. CI, CII and VI,
126 VII) seems to have biological relevance and preserve the ability to create the hair-like projections on the cell surface and the motility defect observed in NAD 47/89
114. In addition, LSP1 has also several serine and threonine residues that can be phosphorylated by kinases like MAPKAPK2 (MK2) or protein kinase C (PKC) 109, 110,
115-118, which are essential for chemotaxis regulation of immune cells.
Extending earlier data from dendritic cells 88, we found LSP1 not only colocaliz-ing with podosome cores, but decoratcolocaliz-ing especially the podosome cap and lat-eral/interconnecting actin filaments. Moreover, LSP1 was significantly enriched at the cell cortex and at the leading edge of migrating macrophages, where it prefer-entially localized at precursor rather than successors podosomes, pointing to a po-tential role in leading edge stabilization and migration.
Further confirmation for this hypothesis came from siRNA-mediated knockdown experiments, where depletion of LSP1 resulted in several effects on multiple levels, influencing individual podosomes, clusters of podosomes and overall cell dynam-ics. In particular, a reduction of about 50 % of protein levels is sufficient to shorten podosome lifetime and induce formation of multiple and highly dynamic clusters of podosomes that randomly move within the cell (Figure 12).
Usually, macrophages have only one cluster of podosomes which can be either in a dynamic steady state (i.e. resting cell with podosomes covering the whole ventral area) or move together with the leading edge of migrating cells, contributing to its stabilization (Figure 12).
Figure 12. LSP1 depletion leads to enhanced mobility of podosome clusters 53. Sequential frames from time lapse videos were progressively color-coded along the spec-trum and merged in a single image. As consequence, static objects tend to be white, whereas moving objects acquire a rainbow-like pattern. To note, in (a) a typical resting cell (up-right) in comparison to a typical migrating macrophage moving along a vertical axis. Rainbow-colored podosome clusters in (b) and (c) indicate enhanced mobility.
127 Depletion of LSP1 destabilizes the whole podosome network and impairs the for-mation of a functional leading edge, producing a phenotype characterized by mac-rophages moving faster, but randomly, with formation of multiple leading edges. In addition, it is conceivable that reduction of LSP1 at the cell cortex can induce re-laxation of cortical tension and improve the ability of the cell to deform and squeeze through ECM pores thus contributing to increase cell adhesion area and collagen I invasion observed in 2D and 3D settings, respectively.
These observations are in line with previous work, where leukocytes from LSP1-knockout mice show faster migration 119, whereas overexpression of the protein, typical of the neutrophil actin dysfunction syndrome (NAD47/89), leads to re-duced motility of neutrophils 113, thus pointing to LSP1 as a negative regulator of immune cell migration.
As mentioned earlier, LSP1 localizes on top of the podosome core, i.e. the cap structure, and partially decorates unbranched actin fibers that extend along its side (i.e. lateral cables), connecting the actin core to adhesion proteins of the ring.
These bundles of F-actin are also sites of myosin IIA localization, which allows them to contract 120. The contractility of lateral cables together with actin polymer-ization, are responsible for the mechanosensing ability of podosomes, allowing them to oscillate perpendicular to the ventral plasma membrane and to exert a protruding force against it, in the range of several nN 75, 121, 122.
Consistent with these observations, we measured an increase in podosome oscilla-tion activity upon LSP1 overexpression, whereas its depleoscilla-tion resulted in 50% less myosin IIA around the podosome core, with concomitant reduction of protrusion forces exerted by single podosomes, as measured by atomic force microscopy (AFM) (Figure 13).
Figure 13. Principle of atomic force mi-croscopy for measuring podosome protru-sive forces 53.
Macrophage is seeded on a pliant sub-strate (Formvar) and turned upside down to allow a cantilever to probe protruding forces exerted by single podosomes.
128 From previous studies, LSP1 is known to have the ability to directly bind myosins, such as myosin 1e 111, however this does not seem to be the case with myosin IIA, where the interaction is mediated by F-actin, as we demonstrated by myosin im-munoprecipitation experiments in the presence of Mg2+ /ATP, which reduce the amount of coprecipitated F-actin with concomitant reduction of LSP1, and myosin cosedimentation assays with pure proteins. We can therefore reason that deple-tion of LSP1 leads to concomitant reducdeple-tion of myosin IIA, both at podosomes and the cell cortex, by decreasing the amount of F-actin bundles and, as a consequence, the number of myosin IIA molecules recruited.
In conclusion, we describe a new key role for LSP1 in regulating the mechanosens-ing activity of podosomes by ensurmechanosens-ing the correct functionality of podosome lateral actin fibers. In addition, impairment of LSP1 activity is not only affecting dynamics of single podosomes, but also alters the stability of the whole podosome network and, in consequence, the overall migratory capability of macrophages.
3. LSP1 competes with supervillin for F-actin and myosin IIA