Nanobody-induced perturbation of LFA-1/L-plastin phosphorylation impairs MTOC docking, immune synapse formation and T cell activation

Clercq, Sarah De; Zwaenepoel, Olivier; Martens, Evelien; Vandekerckhove, Joël; Guillabert, Aude; Gettemans, Jan

doi:10.1007/s00018-012-1169-0

Cited by 42 publications

(60 citation statements)

References 39 publications

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“…This was further confirmed by proteomic analysis of macrophage podosome fractions (54). Therefore, further research mainly focused on the role of L-plastin in podosomes and immune cell function (27)(28)(29). The ubiquitous T-plastin on the other hand has been reported to be a component of cancer cell invadopodia, although siRNA only affected invadopodium length, not their number or matrix degradation capacity (9).…”

Section: Fascin Guarantees Protrusion and Structural Rigidity Whereamentioning

confidence: 78%

Fascin Rigidity and L-plastin Flexibility Cooperate in Cancer Cell Invadopodia and Filopodia

Audenhove¹,

Denert²,

Boucherie³

et al. 2016

Journal of Biological Chemistry

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Invadopodia and filopodia are dynamic, actin-based protrusions contributing to cancer cell migration, invasion, and metastasis. The force of actin bundles is essential for their protrusive activity. The bundling protein fascin is known to play a role in both invadopodia and filopodia. As it is more and more acknowledged that functionally related proteins cooperate, it is unlikely that only fascin bundles actin in these protrusions. Another interesting candidate is L-plastin, normally expressed in hematopoietic cells, but considered a common marker of many cancer types. We identified L-plastin as a new component of invadopodia, where it contributes to degradation and invasiveness. By means of specific, high-affinity nanobodies inhibiting bundling of fascin or L-plastin, we further unraveled their cooperative mode of action. We show that the bundlers cannot compensate for each other due to strikingly different bundling characteristics: L-plastin bundles are much thinner and less tightly packed. Composite bundles adopt an intermediate phenotype, with fascin delivering the rigidity and strength for protrusive force and structural stability, whereas L-plastin accounts for the flexibility needed for elongation. Consistent with this, elevated L-plastin expression promotes elongation and reduces protrusion density in cells with relatively lower L-plastin than fascin levels.Filopodia are finger-like protrusions coordinating movement at the cell front by "sensing" extracellular stimuli (1). Invadopodia on the other hand are formed at the ventral cell side, enabling matrix degradation by localized proteolysis (2). As both actin-based protrusions contribute to migration and invasion, they are associated with cancer metastasis (3, 4), which has also been shown in vivo (5, 6).The architecture and dynamics of filopodia and invadopodia are controlled by a broad variety of actin-binding proteins (7,8). Moreover, these regulating proteins show highly similar spatial and temporal distributions in filopodia and invadopodia (9).

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Section: Fascin Guarantees Protrusion and Structural Rigidity Whereamentioning

confidence: 78%

Fascin Rigidity and L-plastin Flexibility Cooperate in Cancer Cell Invadopodia and Filopodia

Audenhove¹,

Denert²,

Boucherie³

et al. 2016

Journal of Biological Chemistry

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“…Inhibitory conformation-sensitive nanobodies were used to investigate the contribution of L-plastin to the formation of immune synapse (56). L-plastin is an actin-binding protein, which redistributes to the immune synapse following interaction between the T cells and the antigen-presenting cells.…”

Section: Nanobodies Capture Transient Protein Conformationsmentioning

confidence: 99%

Nanobodies as Probes for Protein Dynamics in Vitro and in Cells

Dmitriev

Lutsenko

Muyldermans

2016

Journal of Biological Chemistry

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Nanobodies are the recombinant antigen-recognizing domains of the minimalistic heavy chain-only antibodies produced by camels and llamas. Nanobodies can be easily generated, effectively optimized, and variously derivatized with standard molecular biology protocols. These properties have triggered the recent explosion in the nanobody use in basic and clinical research. This review focuses on the emerging use of nanobodies for understanding and monitoring protein dynamics on the scales ranging from isolated protein domains to live cells, from nanoseconds to hours. The small size and high solubility make nanobodies uniquely suited for studying protein dynamics by NMR. The ability to produce conformation-sensitive nanobodies in cells enables studies that link structural dynamics of a target protein to its cellular behavior. The link between in vitro and in-cell dynamics, afforded by nanobodies, brings the analysis of such important events as receptor signaling, membrane protein trafficking, and protein interactions to the next level of resolution.

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“…[20][21][22][23]30 In this study, detailed insight into the proteolytic gelsolin amyloidosis cascade 12,13 enabled us to target the C68 precursor that gives rise to amyloidogenic peptides. Preventing C68 proteolysis by MT1-MMP may embody a therapeutically preferred strategy since metalloproteases participate in many diverse physiological processes.…”

Section: Discussionmentioning

confidence: 99%

“…19 Our recent work has shown that they can be instrumental in preventing breast cancer metastasis in vivo 20 or perturb selected functions of proteins when used as intrabody (protein domain knockout). [21][22][23][24] In this study, we used nanobodies against the 8 kDa gelsolin peptide to unsettle MT1-MMP proteolysis, which plays an essential role in the gelsolin amyloidosis pathological cascade. MT1-MMP is a membrane bound protease, member of the matrix-metalloprotease (MMP) family.…”

Section: Introductionmentioning

confidence: 99%

Chaperone Nanobodies Protect Gelsolin Against MT1-MMP Degradation and Alleviate Amyloid Burden in the Gelsolin Amyloidosis Mouse Model

et al. 2014

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Gelsolin amyloidosis is an autosomal dominant incurable disease caused by a point mutation in the GSN gene (G654A/T), specifically affecting secreted plasma gelsolin. Incorrect folding of the mutant (D187N/Y) second gelsolin domain leads to a pathological proteolytic cascade. D187N/Y gelsolin is first cleaved by furin in the trans-Golgi network, generating a 68 kDa fragment (C68). Upon secretion, C68 is cleaved by MT1-MMP-like proteases in the extracellular matrix, releasing 8 kDa and 5 kDa amyloidogenic peptides which aggregate in multiple tissues and cause disease-associated symptoms. We developed nanobodies that recognize the C68 fragment, but not native wild type gelsolin, and used these as molecular chaperones to mitigate gelsolin amyloid buildup in a mouse model that recapitulates the proteolytic cascade. We identified gelsolin nanobodies that potently reduce C68 proteolysis by MT1-MMP in vitro. Converting these nanobodies into an albumin-binding format drastically increased their serum half-life in mice, rendering them suitable for intraperitoneal injection. A 12-week treatment schedule of heterozygote D187N gelsolin transgenic mice with recombinant bispecific gelsolin-albumin nanobody significantly decreased gelsolin buildup in the endomysium and concomitantly improved muscle contractile properties. These findings demonstrate that nanobodies may be of considerable value in the treatment of gelsolin amyloidosis and related diseases.

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Nanobody-induced perturbation of LFA-1/L-plastin phosphorylation impairs MTOC docking, immune synapse formation and T cell activation

Cited by 42 publications

References 39 publications

Fascin Rigidity and L-plastin Flexibility Cooperate in Cancer Cell Invadopodia and Filopodia

Fascin Rigidity and L-plastin Flexibility Cooperate in Cancer Cell Invadopodia and Filopodia

Nanobodies as Probes for Protein Dynamics in Vitro and in Cells

Chaperone Nanobodies Protect Gelsolin Against MT1-MMP Degradation and Alleviate Amyloid Burden in the Gelsolin Amyloidosis Mouse Model

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