Invadopodia are actin-rich protrusions arising through the orchestrated regulation of precursor assembly, stabilization, and maturation, endowing cancer cells with invasive properties. Using nanobodies (antigen-binding domains of Camelid heavy-chain antibodies) as perturbators of intracellular functions and/or protein domains at the level of the endogenous protein, we examined the specific contribution of fascin and cortactin during invadopodium formation in MDA-MB-231 breast and PC-3 prostate cancer cells. A nanobody (K(d)~35 nM, 1:1 stoichiometry) that disrupts fascin F-actin bundling emphasizes the importance of stable actin bundles in invadopodium array organization and turnover, matrix degradation, and cancer cell invasion. Cortactin-SH3 dependent WIP recruitment toward the plasma membrane was specifically inhibited by a cortactin nanobody (K(d)~75 nM, 1:1 stoichiometry). This functional domain is shown to be important for formation of properly organized invadopodia, MMP-9 secretion, matrix degradation, and cancer cell invasion. Notably, using a subcellular delocalization strategy to trigger protein loss of function, we uncovered a fascin-bundling-independent role in MMP-9 secretion. Hence, we demonstrate that nanobodies enable high resolution protein function mapping in cells.

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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Diffusion magnetic resonance imaging biomarkers can provide quantifiable information of the brain tissue after a mild traumatic brain injury (mTBI). However, the commonly applied diffusion tensor imaging (DTI) model is not very specific to changes in the underlying cellular structures. To overcome these limitations, other diffusion models have recently emerged to provide a more complete view on the damage profile following TBI. In this study, we investigated longitudinal changes in advanced diffusion metrics following experimental mTBI, utilising three different diffusion models in a rat model of mTBI, including DTI, diffusion kurtosis imaging and a white matter model. Moreover, we investigated the association between the diffusion metrics with histological markers, including glial fibrillary acidic protein (GFAP), neurofilaments and synaptophysin in order to investigate specificity. Our results revealed significant decreases in mean diffusivity in the hippocampus and radial diffusivity and radial extra axonal diffusivity (RadEAD) in the cingulum one week post injury. Furthermore, correlation analysis showed that increased values of fractional anisotropy one day post injury in the hippocampus was highly correlated with GFAP reactivity three months post injury. Additionally, we observed a positive correlation between GFAP on one hand and the kurtosis parameters in the hippocampus on the other hand three months post injury. This result indicated that prolonged glial activation three months post injury is related to higher kurtosis values at later time points. In conclusion, our findings point out to the possible role of kurtosis metrics as well as metrics from the white matter model as prognostic biomarker to monitor prolonged glial reactivity and inflammatory responses after a mTBI not only at early timepoints but also several months after injury.