Force across metavinculin tension sensor (MTS) seems to be less than across vinculin tension sensor (VTS). This effect was especially pronounced on elastic soft substrates (ESS) and vanished upon expression in cell lacking both talin-1and talin-2(Fig. 6.18A & Fig. 6.19A). Several mechanisms have the potential to modulate force transduction across metavinculin (Fig. 7.2B-D):
• Metavinculin could bind VBSs on talin independent from engagement with the actin
cytoskeleton.
• Insertion of metavinculin molecules could lead to a reduction of force by distributing
it across multiple proteins.
• Direct interaction between metavinculin tail domain and F-actin could destabilize
filaments.
A
B
D
C
force transduction across vinculin metavinculin binding to talin without cytoskeletal engagement
distribution of force across multiple metavinculin molecules
mechanical destabilization of actin by metavinculin tail domain
Figure7.2: Models explaining reduced force across metavinculin A.Vinculin (green) is bound to both talin (gray) and the F-actin cytoskeleton (red). The domains of talin which are reoriented upon binding to vinculin and F-actin are shown in yellow.B.-D.depict models how reduced force across metavinculin (orange) could be explained: B.Metavinculin head domain bind talin without engaging to the F-actin cytoskeleton, thus blocking cytoskeletal linkages and reducing force. C.Parallel insertion of metavinculin molecules leads to a reduced mean force across the proteins. D.The MVt insert affects actin stability and reduces the force which can be beared by the linkage. This mechanism could be mediated by severing as well as changes in actin persistence length or bundling properties.
7.3 Factors contributing to force transduction across TS constructs
Using various TS constructs I tried to distinguish between the contribution of different factors to the force transduction across both VTS and MTS. In the following section, I will discuss, how expression of additional protein, engagement with the F-actin cytoskeleton, and altered HTI contribute to the modulation of mechanical force across vinculin and metavinculin.
7.3.1
Total amount of protein and turnover rates
Expression levels of vinculin and metavinculin show complex regulation, whichin vivocan be modulated by factors as different as development, pregnancy, application of external forces or disease (see1.3.5). In flies it has also been shown that overexpression of vinculin influences cardiac architecture and increases the life span of animals [97].
In the fibroblast model I mimicked the effect of different amount of protein using co- transfection of constructs in a modified pLPCX (pLPCXmod) expression vector system. Western blots confirmed that the expression level of VV or MV constructs were comparable to vinculin levels of floxed (vincf/f) cells (Fig. 6.3 A).
FRAP experiments show that increasing the amount of protein leads to faster turnover rates and higher mobile fractions for both vinculin and metavinculin but the difference between the isoforms is even more pronounced and the effect on metavinculin turnover is weaker (Fig. 6.6). Faster turnover can be explained by faster re-binding of protein to unoccupied binding sites, e.g. on talin molecules. It is important to keep in mind that talin binding is important for (meta)vinculin localization at FA, but other binding partners like PIP2 and
unbound molecules also contribute to the observed FRAP signal.
The TS experiments show that force across VTS increases upon expression of additional vinculin or metavinculin, whereas force across MTS does not seem to be affected (Fig. 6.20
B). From immunoprecipitation experiments we know that 5-times more metavinculin is bound to talin-1(Fig. 6.9A & B). This suggests that there is still a large potential for vinculin to occupy additional binding sites on talin-1.
Data from single-molecule experiments lead to the conclusion that vinculin binds to talin in a cooperative manner and one binding triggers a series of other events [136]. Therefore a relatively moderate increase of vinculin concentration could already have a large effect on protein recruitment. It is also known, that once vinculin binds to talin-1and is actively engaged with the actin cytoskeleton, previously cryptic VBSs open up [31, 32].
On the other hand, metavinculin binding to talin-1 could already be close to saturation and therefore might not be affected by expression of additional protein. However, it seems
7.3 Factors contributing to force transduction across TS constructs
that the presence of metavinculin does not alter the amount of force transduced across VTS compared to additional vinculin.
7.3.2
Association with the F-actin cytoskeleton
Introducing mutations into TS constructs enabled me to further understand how actin binding contributes to force transduction. The actin binding mutation I997A, which has been first described by [27], dramatically decreased force across VTS. An additional experiment to test the effects of F-actin binding on force across VTS would be mutation V1001A, which has been described to have an intermediate affinity [78].
Since the beginning of this thesis, two major contributions have been made by others to our understanding of how the metavinculin tail insert affects F-actin organization: in2012
Janssen et al. [79] proposed that metavinculin promotes actin severing, however three years later this view was challenged by the observation that actin fragmentation does not occur in real time assays [80]. To date the most convincing conclusion from in vitrostudies is that metavinculin results in a2-fold reduced actin persistence length lp, but does not lead to the
sufficient structural changes to sever F-actin [80].
In immunostainings I did not observe changes in the global F-actin structure for cells expressing VV or MV, however, this does not exclude local changes. Measurement of the actin dynamics was beyond the scope of this thesis but in accordance with the molecular clutch hypothesis my prediction is that metavinculin is not able to slow down the retrograde flow of F-actin as much as vinculin does [27].
Comparison to other actin severing proteins reveals a 47 % identity between gelsolin and the metavinculin insert from position 936-950[79]. This region also contains the actin binding motif AAIVQLDDYL [132]. Interestingly, there is a gain of function mutation, which enables the only non-severing member of the gelsolin family CapG to sever actin [133]. I used this information about the CapG amino acid sequence to design my own metavinculin mutant. Forces across MTS increased upon change of DDY to the non-severing NTLmotif from the CapG amino acid sequence, indicating the importance of this region for mechanotransduc- tion. This mutation of metavinculin has not been described in the literature before and has the potential to contribute to our understanding of fundamental differences between active severing and induction of mechanical discontinuities leading to actin fragmentation. This is also crucial to understand other severing molecules like ADF/cofilin [80]. Cryo-electron mi- croscopy (EM) on the lamellipodia of VV and MV or superresolution microscopy could give insight into the size distribution and bending of actin filaments close to FA. In combination
7.3 Factors contributing to force transduction across TS constructs
with actin speckle miscroscopy, the relevance of metavinculin and especially the DDY motif for F-actin bundling might further be elucidated.
7.3.3
Effects of the HTI on force transduction
The HTI mutations severly affected FA and cell morphology for both vinculin and metavin- culin, indicating large scale effects on FA and cytoskeletal organization. I observed hyper- trophic FA, which were not able to disassemble and some cells showed protein accumula- tions.
While it is widely accepted that vinculin activation state is important for its localization and function at FA [25], it was not clear how the HTI interaction affects force transmission. I can state that force increases across both VTS and MTS when HTI is lowered (Fig. 6.21). This is the only experiment in which we observe FRET efficiency values for the MTS between 5−10 %, which shows that it is indeed possible to increase the mean force across MTS. Similar to vinculin overexpression, increasing the amount of active vinculin protein could lead to a reinforcing effect caused by cooperative binding to talin.
7.3.4
Implications of reduced force transduction across metavinculin
Metavinculin expression could either provide resistance against external mechanical stimuli or uncouple the acto-myosin machinery from adhesion sites. It is stabilized at FA and could therefore also increase the stability of the talin scaffold and thus the remaining adhesion site.
Evidence accumulates that metavinculin indeed uncouples adhesion plaques from the cytoskeleton. This could either prevent high actomyosin generated forces to tear apart the adhesion or limit the size and strength of actin bundles, which could otherwise inhibit disassembling of adhesions and ultimately cause cells to be incapable of remodelling and migration.