Actin Turnover in Lamellipodial Fragments.

Actin turnover is the central driving force underlying lamellipodial motility. The molecular components involved are largely known, and their properties have been studied extensively in vitro. However, a comprehensive picture of actin turnover in vivo is still missing. We focus on fragments from fish epithelial keratocytes, which are essentially stand-alone motile lamellipodia. The geometric simplicity of the fragments and the absence of additional actin structures allow us to characterize the spatiotemporal lamellipodial actin organization with unprecedented detail. We use fluorescence recovery after photobleaching, fluorescence correlation spectroscopy, and extraction experiments to show that about two-thirds of the lamellipodial actin diffuses in the cytoplasm with nearly uniform density, whereas the rest forms the treadmilling polymer network. Roughly a quarter of the diffusible actin pool is in filamentous form as diffusing oligomers, indicating that severing and debranching are important steps in the disassembly process generating oligomers as intermediates. The remaining diffusible actin concentration is orders of magnitude higher than the in vitro actin monomer concentration required to support the observed polymerization rates, implying that the majority of monomers are transiently kept in a non-polymerizable "reserve" pool. The actin network disassembles and reassembles throughout the lamellipodium within seconds, so the lamellipodial network turnover is local. The diffusible actin transport, on the other hand, is global: actin subunits typically diffuse across the entire lamellipodium before reassembling into the network. This combination of local network turnover and global transport of dissociated subunits through the cytoplasm makes actin transport robust yet rapidly adaptable and amenable to regulation.

Differential mapping of the free barbed and pointed ends of actin filaments in cells.

The actin cytoskeleton plays a pivotal role in many cellular processes. Detailed analysis of the architecture of cellular actin networks provides valuable insight into the dynamic self-organization underlying these processes. In particular, since most of the actin turnover occurs at the tips of actin filaments, it is insightful to map the distribution of filament ends. Here we report a method for differentially labeling the pointed and the barbed ends of actin filaments in cellular networks by permeabilizing cells, following a brief fixation, and introducing labeled actin monomers in the presence or absence of capping protein, respectively. This method quantitatively maps the distributions of free barbed ends and free pointed ends in adherent cells, providing information on the polarity of cytoskeletal structures and mapping active sites available for actin assembly or disassembly. We demonstrate the use of this method by mapping the distribution of actin filament ends in motile fish epithelial keratocytes and in several mammalian cell lines, and show that free barbed ends are enriched near the tip of protruding lamellipodia while free pointed ends concentrate toward the rear.

Actin disassembly clock determines shape and speed of lamellipodial fragments.

A central challenge in motility research is to quantitatively understand how numerous molecular building blocks self-organize to achieve coherent shape and movement on cellular scales. A classic example of such self-organization is lamellipodial motility in which forward translocation is driven by a treadmilling actin network. Actin polymerization has been shown to be mechanically restrained by membrane tension in the lamellipodium. However, it remains unclear how membrane tension is determined, what is responsible for retraction and shaping of the rear boundary, and overall how actin-driven protrusion at the front is coordinated with retraction at the rear. To answer these questions, we utilize lamellipodial fragments from fish epithelial keratocytes which lack a cell body but retain the ability to crawl. The absence of the voluminous cell body in fragments simplifies the relation between lamellipodial geometry and cytoskeletal dynamics. We find that shape and speed are highly correlated over time within individual fragments, whereby faster crawling is accompanied by larger front-to-rear lamellipodial length. Furthermore, we find that the actin network density decays exponentially from front-to-rear indicating a constant net disassembly rate. These findings lead us to a simple hypothesis of a disassembly clock mechanism in which rear position is determined by where the actin network has disassembled enough for membrane tension to crush it and haul it forward. This model allows us to directly relate membrane tension with actin assembly and disassembly dynamics and elucidate the role of the cell membrane as a global mechanical regulator which coordinates protrusion and retraction.

The quadruplex r(CGG)n destabilizing cationic porphyrin TMPyP4 cooperates with hnRNPs to increase the translation efficiency of fragile X premutation mRNA.

The 5' untranslated region of the FMR1 gene which normally includes 4-55 d(CGG) repeats expands to > 55-200 repeats in carriers of fragile X syndrome premutation. Although the levels of premutation FMR1 mRNA in carrier cells are 5-10-fold higher than normal, the amount of the product FMR protein is unchanged or reduced. We demonstrated previously that premutation r(CGG)(n) tracts formed quadruplex structures that impeded translation and lowered the efficiency of protein synthesis. Normal translation could be restored in vivo by the quadruplex r(CGG)(n) destabilizing action of CBF-A and hnRNP A2 proteins. Here we report that the quadruplex-interacting cationic porphyrin TMPyP4 by itself and in cooperation with CBF-A or hnRNP A2 also unfolded quadruplex r(CGG)(n) and increased the efficiency of translation of 5'-(CGG)(99) containing reporter firefly (FL) mRNA. TMPyP4 destabilized in vitro a (CGG)(33) intramolecular quadruplex structure and enhanced the translation of 5'-(CGG)(99)-FL mRNA in a rabbit reticulocyte lysate and in HEK293 cells. The efficiency of translation of (CGG)(99)-FL mRNA was additively increased in cells exposed to TMPyP4 together with CBF-A. Whereas low doses of TMPyP4, CBF-A or hnRNP A2 by themselves did not affect the in vivo utilization of (CGG)(99)-FL mRNA, introduction of TMPyP4 together with either protein synergistically augmented its translation efficiency.