Dysferlin regulates cell adhesion in human monocytes

Dysferlin is mutated in a group of muscular dystrophies commonly referred to as dysferlinopathies. It is highly expressed in skeletal muscle, where it is important for sarcolemmal maintenance. Recent studies show that dysferlin is also expressed in monocytes. Moreover, muscle of dysferlinopathy patients is characterized by massive immune cell infiltrates, and dysferlin-negative monocytes were shown to be more aggressive and phagocytose more particles. This suggests that dysferlin deregulation in monocytes might contribute to disease progression, but the molecular mechanism is unclear. Here we show that dysferlin expression is increased with differentiation in human monocytes and the THP1 monocyte cell model. Freshly isolated monocytes of dysferlinopathy patients show deregulated expression of fibronectin and fibronectin-binding integrins, which is recapitulated by transient knockdown of dysferlin in THP1 cells. Dysferlin forms a protein complex with these integrins at the cell membrane, and its depletion impairs cell adhesion. Moreover, patient macrophages show altered adhesion and motility. These findings suggest that dysferlin is involved in regulating cellular interactions and provide new insight into dysferlin function in inflammatory cells.

2 Compartment Myopathy (MIM#606768) (3), collectively referred to as dysferlinopathies. All of them are characterized by an adult onset of muscle weakness, followed by progressive muscle wasting. The muscle tissue presents with strong inflammatory infiltrates (4), and for that reason the disease is often misdiagnosed for the immune disorder polymyositis. Dysferlin is a C2 domain containing transmembrane protein that is highly expressed in skeletal muscle (5) and activated satellite cells (6), and increases with differentiation. In addition it is expressed in monocytes (7,8). Recent studies suggest that the immune response in dysferlin deficient tissue is compromised (9,10). In muscle, dysferlin deficiency causes reduced cytokine (9,10) and chemokine (11) secretion and a subsequent local misbalance in recruitment and persistence of immune cells (9). In dysferlin mouse models, neutrophils and macrophages appear later at the site of damage compared to controls in degeneration/regeneration experiments (9). Yet after their late appearance these cells reside longer in the muscle tissue, suggesting a prolonged inflammatory response of these cells (9). Roche et al (12) showed the existence of a strong immune component in dysferlin deficiency that contributes to its slower recovery from injury As dysferlin is strongly expressed in monocytes (7,8), it was suggested that modified monocyte behavior might contribute to the dysferlin phenotype. Indeed a recent study indicates that dysferlin deficient macrophages have a higher phagocytosis index and are thus more aggressive than wild-type cells (13). However, the mechanism behind this observation is unclear.
We previously characterized the dysferlin protein complex in muscle and observed that dysferlin is found in complex with focal adhesion components in skeletal muscle cells (14). Focal adhesions are cellular attachment sites where the internal cytoskeleton is connected to the extracellular matrix via integrins (15).
Integrins are heterodimeric transmembrane receptors that consist of an α-and a βsubunit and are central to focal adhesions (15). According to the traditional view integrins link the extracellular matrix to the intracellular cytoskeleton (15,16). However, recent studies also indicated a role for integrins in cell-cell contacts (16). Because of dysferlin's uncharacterized role near focal adhesions and the deregulated monocyte response in dysferlinopathy patients we investigated a potential function for dysferlin in monocyte cell adhesion. We observed a striking increase in macrophage motility in dysferlinopathy patients, in line with an altered adhesion propensity.
Western blot detection -Cells were counted, dissolved in sample buffer and boiled for 10 min. Resulting protein homogenates were separated on SDS-page gels and transferred onto nitrocellulose (for AHNAK) or PVDF membranes. Protein loading was standardized to cell count and monitored with Ponceau S staining of the blot directly after transfer. After antibody detection blots were analyzed with an Odyssey scanner (Licor). Protein bands were quantified with ImageJ, and Odyssey analysis software.
RNA isolation and cDNA synthesis RNA was extracted with a RNA extraction kit (Macherey-Nagel) according to manufacturer's protocol. Subsequent cDNA synthesis was performed with a kit and random hexamer primers (Fermentas) according to manufacturer's instructions. 1µg RNA was used as input for cDNA reactions.
Quantitative RT-PCR -All primers were designed with the webtool Primer3 (URL), with mispriming against human database. Quantitative PCR reactions were performed with SYBRgreen, in 15µl reaction volume with 3ng cDNA input. All primers had comparable efficiencies between 95% and 105%. All measurements were performed in triplo. Statistical analysis was performed according to the method Pfaffl (18). GAPDH was used as a reference gene.
DNA plasmids and transfection -The following shRNA plasmids out of the Sigma Mission shRNA library were used in this study: non-target SHC002, and Dysferlin TRCN0000000967. The Dysferlin shRNA was validated by co-expression with Dysferlin expression vector. 10^6 THP1 cells were washed in PBS and dissolved in nucleofactor buffer V. 0.5µg DNA plasmid was added. Transfection was achieved by electroporation with an Amaxa Nucleofactor device. Transfected cells were resuspended in culture medium.
Immunostaining -Differentiated THP1 cells were fixed in formalin solution for 10 min, following by permeabilization in 0.3% Triton (Sigma) for 5 min. Cells were washed, blocked in 1% BSA (Sigma) for 10 min, and incubated with 1st antibody 2h RT. Cells were subsequently washed 3x and incubated with 2nd antibody for 1h RT. Cells were washed in PBS and mounted onto slides with AquaPolymount supplemented with DAPI. For the visualization of the very transient focal adhesions we used CSK buffer. THP1 cells were differentiated at coverslips for >3days using PMA. Cells were fixed with fresh CSK buffer (0.3% Triton, 10mM Pipes pH 7.0, 50mM KCl, 2mM CaCl2, 2mM MgCl2, 300mM sucrose, 4% paraformaldehyde) for 15min. This treatment washed out all of the soluble cell components but fixes the cytoskeletal parts including the focal adhesions. The coverslips were subsequently washed 2x in PBS and blocked with 1% BSA in PBS for >10 min. Next the coverslips were stained with the antibodies as mentioned above. For the endocytosis assays, cells were incubated for 1h on ice with ITGB3 antibody at 1;10,000. Nonbound antibody was washed of with PBS, and fresh medium was added. Cells were cultured at 37°C for 1h. Endocytosis was stopped by putting the cells on ice. Surface antibody was washed of with 0.2 mM glycine (pH 2.5). Next cells were prepared as otherwise. As a control, cells were analyzed directly after antibody incubation, confirming that all surface-bound antibody could be washed of by the glycine treatment (not shown). To inhibit the Integrins differentiated THP1 cells were incubated in 1µg/ml GRGDS (Sigma), or a control peptide (SDGRG, Sigma). For the ITGB3 stimulation cells were incubated with ITGB3 antibody at 37°C.
Immunoprecipitation -To obtain total cell lysates differentiated THP1 cells were lysed by scraping in triton buffer (50mM TrisHCl, pH 7.5, 150mM NaCl, 0.2% Triton X100, 1x protease inhibitor cocktail (Roche)) after a PBS wash. Cultured cells were prepared freshly by washing in PBS and lysed by scraping on ice in lysis buffer. All homogenates were spun down at maximum speed, 4°C, 20 min. To generate subcellular fractions, differentiated THP1 cells were trypsinized and washed with PBS. The pellet was lysed in sucrose buffer (320 mM Sucrose, 5 mM HEPES (pH 7.4), 5 mM EDTA, 2x Protease inhibitor cocktail) with a dounce homogenizer. Lysates were spun down subsequently at 1,000g (P1, containing insoluble debris), 10,000g (P2, containing mitochondria and peroxisomes) and 100,000g (P3, enriched for cell membrane, endoplasmic reticulum and vesicles (microsomes)). Pellet fractions were dissolved in triton buffer for further immunoprecipitations. Protein A sepahrose CL-4B (GE Healthcare) was washed 3x in lysisbuffer and used to preclear the homogenates for 1h, at 4°C tumbling. Sepharose was removed and antibody added (50µg HCAb) for O/N incubation at 4°C, tumbling. Thereafter washed sepharose was added and incubated for 2h, 4°C, tumbling. Homogenates were spun down at 500g and supernatant stored as non-bound fraction. The sepharose was washed 5x 3x short, 2x long (>20min tumbling at 4°C). Finally, all fluid was removed and protein eluted by boiling in sample buffer.
Macrophages culture and video and immunocytochemistry -Blood samples from 5 patients and 5 healthy controls were extracted using vacutainer CPT tubes (Becton Dickinson, Basel, Switzerland). CD14+ monocytic cells were separated using the negative selection purple EasySep cell selection system (Stemcell technologies, Grenoble, France). Monocytes were differentiated into macrophages during 6 days in culture media based on RPMI 1640 supplemented with 10% FBS, 1% glutamine, 1% sodium pyruvate, penicillinstreptomycin and fungizone and amino acids in poly-D-lysin precoated Lumox plates (Sartedt, Nümbrecht, Germany). Macrophages were filmed in a Laser Scanning Confocal Inverted Microscope Leica TCS SP5-AOBS (Leica Microsystems, Wetzlar, Germany) during 18h at 37ºC and 5% CO2. The videos were analyzed using Leica LAS AF software. In the end we were able to gather data on 29 macrophages from 5 dysferlinopathy patients and 17 macrophages from 5 controls. The distance ran by each studied cell was measured every 30 minutes. Comparison between control macrophages and dysferlin-negative macrophages was performed using a student t-test. To determine the purity of macrophage cultures cells were fixed in cold methanol for 10 minutes at 4ºC and blocked with Tris buffer saline supplemented with 1% BSA, 1% normal human serum, 0.5% donkey serum and 0.01% Triton X100. A primary monoclonal antibody to CD68 (Dako, Glostrup, Denmark) was incubated for 2h at room temperature and labeled with donkey anti-mouse Alexa Fluor 488 (Invitrogen, Carlsbad CA).

RESULTS
Dysferlin expression is enhanced in differentiating monocytes. Previous reports show that dysferlin is expressed in monocytes and macrophages (7,8,13). To confirm this observation we purified B cells, T cells, and monocytes from freshly isolated peripheral blood mononuclear cells (PBMCs) from healthy donors and investigated dysferlin protein levels on western blot. Dysferlin is expressed only in monocytes, conform prior observations, while its interaction partner AHNAK (19) is expressed in all three cell types ( Figure 1A). We next differentiated freshly isolated monocytes in vitro to a pro-inflammatory (M1) or contra-inflammatory (M2) phenotype, and again tested for dysferlin and AHNAK expression on western blot ( Figure 1B). Both proteins are increased with differentiation in both M1 and M2 macrophages. Interestingly, the monocytelike leukemic THP1 cell model shows a similar increase in dysferlin and AHNAK upon differentiation ( Figure 1B). Finally we measured RNA expression levels of dysferlin and AHNAK in THP1 cells ( Figures 1C and 1D). Again, we found a strong upregulation of dysferlin. AHNAK however, is upregulated to a much smaller extent suggesting that the increase in AHNAK protein levels is achieved through a different mechanism than that for dysferlin. We conclude that dysferlin and AHNAK are increased with monocyte differentiation.
Dysferlin expression is insensitive to cellmatrix contact formation. In skeletal muscle, dysferlin functions as a calciumsensitive membrane repair protein (20). In a THP1 cell scratch-wounding assay dysferlin displayed a calcium dependent recruitment to the lesion site (data not shown), indicating that also in monocytes dysferlin participate in calcium-sensitive membrane repair. However, it is not likely that monocytes suffer from frequent membrane damage as do myofibers, challenging the biological relevance of the membrane repair function of dysferlin in monocytes. We therefore explored potential additional functions of dysferlin. We and others previously reported that dysferlin forms a complex with focal adhesion components, which are important in cell-cell and cell-matrix contacts (14,21). Monocytes require interactions with the extracellular matrix and surrounding cells to exert their function. THP1 cells are nonadherent prior to differentiation and become adherent upon differentiation. We therefore investigated whether dysferlin and AHNAK expression levels are sensitive to cell adherence. We first measured the mRNA expression levels of various matrix and adherence proteins in differentiating THP1 cells ( Figure 2A). This showed dysferlin expression to be concomitantly upregulated with fibronectin and integrins α5, αV, β1 and β3. Interestingly, the integrins α5β1 and αVβ3 have fibronectin binding capacity. We could not detect upregulated expression of other matrix proteins such as laminin ( Figure 2A). This suggests that the THP1 cells adhere to fibronectin. The same genes were upregulated in in vitro differentiated M1 and M2 macrophages ( Figure 2B). We proceeded to test THP1 cell adhesion on protein matrixes. We coated wells with PBS (mock), poly L-lysine, collagen or fibronectin. Poly L-lysine is positively charged and attracts all cells to the surface without adherence, while fibronectin and collagen are matrix proteins. Figure 2C shows that on plastic surfaces dysferlin and AHNAK proteins are only increased upon the induction of differentiation. A similar effect is seen for poly L-lysine. Fibronectin however increases AHNAK protein levels, yet not dysferlin, even in the absence of differentiation. Indeed the cells adhere onto a fibronectin matrix without achieving the spread-out differentiated phenotype (not shown). Dysferlin again only increases upon differentiation. Finally, the collagen matrix has no strong effect on either protein unless the cells were differentiated. We conclude that dysferlin expression does not respond to cell-matrix adhesion, contrary to AHNAK, which is sensitive to fibronectin adherence.

Differentiation
induced dysferlin expression is enhanced in response to cellcell contact formation. We next investigated dysferlin and AHNAK expression in response to cell-cell contacts. We differentiated THP1 cells at increasing cell densities, predicting that this would result in an increased frequency of cell-cell contact formation. Surprisingly, the differentiation-induced dysferlin mRNA expression levels are further increased with increasing cell density, suggesting a positive response to cell-cell contact formation ( Figure 3). The integrins ITGA5, ITGAV, ITGB1 and ITGB3 show the opposite pattern, with expression levels dropping with increasing cell density ( Figure 3A). Nevertheless, apart from their function in cell-matrix contacts, integrins are also important for cell-cell contacts (16,22). The dysferlin interaction partner AHNAK shows an increase in expression similar to dysferlin but followed by a sudden drop at the highest cell density tested. It is not clear why this happens, but given its function in cell interactions(23,24) its increase may be a response to the cell's loss in integrin expression. To verify that dysferlin protein is involved in cell-cell contacts we performed immunofluorescent staining experiments on differentiated THP1 cells. Dysferlin strongly accumulates at cell-cell contacts ( Figure 3B), together with ITGB3. Intriguingly, the proteins AHNAK and ITGB1 are also strongly present at cell-cell contacts (not shown), although their RNA expression levels are insensitive to cell density. This indicates that dysferlin plays a role at cell-cell contacts.

Loss of dysferlin protein results in deregulation of integrin expression.
To obtain more evidence that dysferlin is important for cell adhesion, we transfected THP1 cells with an shRNA plasmid to knock down dysferlin ( Figure 4A+B). We confirmed a substantial reduction of dysferlin protein levels by western blotting ( Figure 4B). We measured RNA ( Figure  4A) and protein (figure 4B) expression levels during differentiation and observed that ITGB1 and ITGB3 and fibronectin are much stronger upregulated in the dysferlin depleted cells, while other markers such as CD11b (ITGAM, or integrin αM) appear unaffected. This shows that integrin mRNA expression is affected by the absence of dysferlin, which is suggestive of a change in cell adhesion properties. We next measured RNA expression levels in a confirmed patient with LGMD2B and a matched control, and observed a strikingly similar deregulation of fibronectin and the fibronectin binding integrins in the patient cells ( Figure 4C). Intriguingly though, in the patient we did not detect an increase in ITGB3, and a decrease in ITGAM. Based on these markers the patients cells resemble more the M2 macrophages as shown in Figure 2B. Finally, we performed cell counts at 4 days post-differentiation of mocktransfected and dysferlin depleted THP1 cells. In wild-type cells, most of the cells adhere to the surface after four days of differentiation. Transfection with a nontarget shRNA plasmid results in a slight decrease in adherent cells, probably due to the transfection procedure. However, depletion of dysferlin results in a strong reduction in cell adhesion ( Figure 4E). We stained shRNA transfected THP1 cells for surface ITGB3. Again, this showed a clear loss of adherent cells, but no difference in surface ITGB3 could be detected between dysferlin depleted and control cells, in the adherent cells. This suggests that the localization of integrins is unaffected by dysferlin depletion, but leaves open the possibility of altered dynamics. We conclude that dysferlin is involved in Integrin mediated THP1 monocyte cell adhesion.
Dysferlin can form a complex with focal adhesion components. In skeletal muscle cells, dysferlin forms a complex with vinculin and associated focal adhesion proteins (14). In order to test whether dysferlin is physically associated with adhesion complexes we performed immunoprecipitation experiments on protein homogenates from differentiated THP1 cells. In addition to total homogenates we also analyzed cellular subfractions enriched for organelles or microsomes ( Figure 5). We used two independent dysferlin-specific heavy chain antibody fragments (VHH) and one non-specific VHH, as described previously (14,25) and analyzed coimmunoprecipitation by western blot. Dysferlin is found in all fractions. Moreover, ITGB3, vinculin, paxillin and β-parvin coimmunoprecipitate with dysferlin from the microsomal fraction, suggesting that as in muscle cells, dysferlin forms a complex with a subset of focal adhesions in THP1 cells. ITGB1 does not co-immunoprecipiate with dysferlin. It should be noted that in the total cell extract no detectable amount of ITGB3 coimmunoprecipitates with dysferlin suggesting that the interaction takes place at the focal adhesion only. Also we observe that the amount of protein that is immunoprecipitated with Dysferlin is modest, suggesting that the interactions are either transient, or concern only a subset of the proteins. To further substantiate the evidence for dysferlin near focal adhesions we performed co-immunofluorescence experiments with an antibody against the focal adhesion protein vinculin ( Figure 5C). Focal adhesions have a rapid turnover.
Therefore we fixed the cells with the stringent CSK buffer. This allowed for detection of vinculin in focal adhesions. Dysferlin partially colocalizes with vinculin at these focal adhesions. This modest colocalization might suggest that the interaction is transient in nature. We conclude that dysferlin can form a protein complex with focal adhesion components in monocytes.

Dysferlin endocytosis in response to
Integrin stimulation. A recent study by Sharma et al (26) suggested a role for Dysferlin in the trafficking of the adhesion molecule PECAM in endothelium. We therefore studied the trafficking of dysferlin and ITGB3 in THP1 cells with coimmunofluorescence staining. We observed that in resting differentiated THP1 cells dysferlin and ITGB1 or ITGB3 do not strongly colocalize ( Figure 6A). This confirms the IP data on whole cell lysates and supports the hypothesis that both proteins are together only at the cell membrane or on endocytic vesicles. We therefore decided to stimulate integrin endocytosis by incubating differentiated THP1 cells with small peptide (GRGDS) containing the matrix binding site for integrin (RGD). This peptide competes for the matrix-binding site. Upon binding the peptide the integrin is endocytosed ( Figure  6B) and it is replaced by exocytosis of integrin molecules from an intracellular storage. We used a low concentration that removed the focal adhesions in the cell protrusions (stained by vinculin in Figure  6B), but prevented the cells from rounding up and detaching, and we stained the cells for dysferlin and ITGB3. Both proteins show a rapid response to GRGDS, and accumulate in a perinuclear compartment ( Figure 6A). This recruitment is specific for dysferlin and ITGB3, as subsequent costaining for dysferlin and AHNAK showed the latter to shift towards the cell periphery ( Figure 6C).
Many integrin heterodimers have the potential to bind to an RGD peptide motif (27), and all these heterodimers might be inhibited by the GRGDS treatment. However, our data thus far suggested that mainly ITGB3 is functionally linked to dysferlin. To test this we incubated cells in the presence of a monoclonal antibody specific for the extracellular part of ITGB3. This resulted in a similar recruitment of both ITGB3 and dysferlin ( Figure 6D), suggesting that the GRGDS induced trafficking of dysferlin is largely caused by ITGB3 inhibition.
To prove that the integrins undergo endocytosis upon GRGDS stimulation we incubated differentiated THP1 cells with the integrin β3 antibody on ice, prior to GRGDS stimulation. Subsequent stimulation with GRGDS at 37°C resulted in intracellular ITGB3 antibody, strongly indicating that the integrins are indeed endocytosed in response to the GRGDS treatment ( Figure 6E). As there is no antibody available that binds to the extracellular domain of dysferlin it is technically not feasible to confirm this in a similar experimental setup for dysferlin. We conclude that dysferlin is co-endocytosed with ITGB3.
Dysferlinopathy macrophages move faster. Our data thus far suggest that dysferlin locates to monocyte cell adhesion sites, and that it is involved in cell interactions. We therefore hypothesized that cell adhesion might be disturbed in monocytes of dysferlinopathy patients. To test this we isolated and cultured human primary macrophages from five molecularly confirmed LGMD2B patients, and five healthy matched control donors. All isolated cells stained positive for the marker CD68, confirming the purity of our cultures ( Figure  7A). We performed live cell microscopy to monitor the cells and record their movements. Strikingly, we observed that dysferlinopathy macrophages are more motile than their healthy counterparts ( Figure 7B, C). Specifically, dysferlinopathy macrophages covered more distance and had a 60% increase in average velocity (13,85µm/h compared to 8.71µm/h for controls, with p=0.00009). These data are consistent with the reduced adhesion in dysferlin depleted THP1 cells, and with a role for dysferlin in regulation of cellular interactions.

DISCUSSION
We report that dysferlin expression is upregulated in differentiating monocytes and the THP1 monocyte cell model. Dysferlin localizes to the cell membrane of monocytes and is rapidly endocytosed with ITGB3 upon inhibition of this integrin. Dysferlin forms a complex with ITGB3 and focal adhesion components at the cell periphery. Moreover, our data indicate that in the absence of dysferlin the regulation of fibronectin binding integrins is disturbed. Dysferlin depletion further enhances the increase in ITGB3 expression. This results in attenuated differentiation and adhesion of these cells, which mimics the loss of adhesion observed in dysferlin depleted HUVEC cells(26). In dysferlinopathy macrophages this reveals itself as increased cell motility. Interestingly, monocytes expressing ITGB3 show increased motility (28) compared to cells without ITGB3.
During the recruitment of mononuclear cells to inflamed tissue, these cells first need to adhere to the endothelial cell and start the process of rolling. They subsequently transmigrate through the endothelial cell layer. And finally, they migrate through the tissue to participate in phagocytosis of apoptotic cells. The attachment of circulating monocytes to endothelial cells is in part mediated by integrins that bind to endothelial VCAM molecules and thereby induce tight interaction between the two cell types(29). Such cell-cell interactions generally increase the levels of integrin expression (15) by positive feedback, thereby gradually increasing adherence to facilitate subsequent transmigration.
Our data suggest that dysferlin-depleted THP1 cells adhere less efficiently, but nevertheless increase expression of integrins. Integrins can be activated by outside-in and by inside-out signaling. Outside-in signaling refers to the process where integrins are activated through adhesion and trigger signaling cascades in the cells. Inside-out signaling denotes relocalization of integrins during for instance motility to provide direction (15). Via outside-in signaling, integrins can affect Erk, JNK and PKB and thereby affect gene expression (15). Moreover, cells have numerous feedback mechanisms that link expression of different adhesion proteins. For example, monocyte cell adhesion results in increase levels of fibronectin (30). Moreover, such feedback loops can be negative, as integrin αVβ6 can compensate for the loss of ITGB1 (31,32). Therefore the increased integrin mRNA expression in the absence of dysferlin may be explained as a compensatory mechanism. In muscle similar negative feedback loops have been described for dysferlin. Dysferlin deficient muscle cells produce increased amounts of IL1β (10) and thrombospondin (11) suggesting that dysferlin inhibits expression of these proteins.
When comparing the mRNA expression levels in dysferlinopathy patient monocytes with dysferlin depleted THP1 cells, the deregulated expression of ITGB1 is consistent, contrary to ITGB3. However, cross-talk between integrins is common and has been reported between ITGB1 and ITGB3 in monocytes. ITGB1 (α5β1) is regulated by ITGB3 (αVβ3) in phagocytosis experiments (33). Crosstalk between αVβ3 and α4β1 regulates monocyte migration on VCAM1 (34). Such crosstalk might explain the apparent discrepancy of the mRNA expression data.
We observed that dysferlin and ITGB3 trafficking is co-regulated. This is consistent with dysferlin's role in the transport of the adhesion protein PECAM in endothelium(26). Moreover, the fact that focal adhesion components are identified in the dysferlin protein complex in both monocytes and myoblasts suggest parallels in dysferlin function between these cells. Indeed, the process of phagocytosis has been implicated to be analogous to endocytosis, and both processes require coordinated restructuring of cell membrane and the cortical actin based cytoskeleton.  (9). This was explained by a deregulated communication from the myofibers to recruit the monocytes (9). Our data however provide an additional explanation. In the absence of dysferlin the monocytes and macrophages show strongly reduced adhesion and increased motility, which might influence the infiltration.
In summary, we have identified a potential involvement of dysferlin in cellular adhesion and thereby a potential mechanism for the deregulation of dysferlin deficient immune cells.

21.
Matsuda, C., Kameyama, K., Tagawa      RGD prevent the integrins from binding to the extracellular matrix and thereby stimulate endocytosis. C) As in A) but now the cells were stained for dysferlin and AHNAK. Only dysferlin is recruited intracellularly. In the control situation cells were incubated with a control peptide containing the inverse amino acid sequence (SDGRG), which does not bind integrins. D) Differentiated THP1 cells were incubated with a monoclonal MaITGB3 antibody and stained for dysferlin and ITGB3. Both proteins are recruited to a perinuclear compartment. E) Integrin β3 is endocytosed upon RGD stimulus. Differentiated THP1 cells were incubated with MaITGB3 at 4°C for 1h. Cells were washed and incubated 30 min at 37°C with RGD. Surface-bound antibody was removed with glycine and cells were stained for internalized ITGB3 and dysferlin. Both proteins accumulate at the perinuclear storage compartment, indicating that ITGB3 is endocytosed.

Figure 7:
LGMD2B monocytes move faster than control cells. A) Macrophage culture stained with DAPI and the macrophage marker CD68. B) Graphic representation of average velocity and standard deviation in µm/h of healthy macrophages (controls) and dysferlin-null macrophages (patients) (p=0.00009). C) Pictures of the 18h tracking drawn by control macrophages (a) and dysferlin-null macrophages (b).