br Results br Discussion To achieve quantitative understandi

Results
Discussion To achieve quantitative understanding of CT-99021 turnover, we utilize rapidly moving lamellipodial fragments that are geometrically simple, structurally homogeneous, and persistent, and measure relevant rates and concentrations in this system. We find that the lamellipodial actin network density is ∼0.8 mM and decreases approximately 2-fold from front to rear (Figure 1E), conforming to earlier studies [3, 17, 31, 45] and suggesting a slow net network disassembly rate. Our results indicate that this net disassembly rate reflects the combined effect of much faster network disassembly and reassembly occurring throughout the lamellipodium. Both FRAP and extraction experiments show that roughly two-thirds of the actin is in the diffusible pool, so the diffusible actin concentration is ∼1.6 mM. Roughly a quarter of this diffusible actin is in filamentous form as short diffusing oligomers (∼400 μM, ∼13 subunits). These results are aligned with earlier work [40, 42, 46], suggesting that the presence of oligomers is a general feature of cellular actin networks. These observations are also consistent with a growing body of studies on the molecular mechanisms of actin network disassembly, which highlight severing and debranching as important steps that generate oligomers as intermediates in the disassembly process [22, 23, 24, 25, 27]. The remaining diffusible actin concentration is still orders of magnitude higher than the in vitro actin monomer concentrations required to support the observed polymerization rates, as noted previously in other cell types [17]. It is thus highly unlikely that most of the diffusible actin in cells is available for polymerization. Thus, we argue that the majority of monomers are transiently kept in a “reserve” pool, unavailable for assembly, perhaps by being sequestered by thymosin. Our results indicate that the actin network turnover is local, whereas the diffusible actin transport is global (Figure 5C). An actin subunit typically travels in the network ∼1 μm before dissociating. The front-to-rear width of the fragment is an order of magnitude longer than this, so every part of the network undergoes ∼10 cycles of reassembly across the lamellipodium. In contrast, 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 (Figure S6; STAR Methods). We note that in the case of larger cells, estimates that led to these conclusions will have to be reexamined.
STAR★Methods
Author Contributions
Acknowledgments We thank Erez Braun and Tzer Han Tan for comments on the manuscript. We thank Gidi Ben Yoseph for technical help and support. We thank Nitsan Dahan from the LS&E Imaging and Microscopy Unit for help with the FRAP and FCS experiments. We thank Liora Garion for her help with some of the experiments. This work was supported by a Levi Eshkol fellowship from the Israel Science Ministry (to N.O.), United States-Israel Binational Science Foundation grant number 2013275 (to K.K. and A.M.), and NIH grant number GM068952 (to A.M.).
Introduction Actin filaments drive many cellular processes such as cell motility, membrane transport, chemotaxis, cellular morphogenesis and force generation [1], [2]. The formation and determination of the correct lengths of actin filaments are essential for many cellular processes to take place in an orderly manner. Actin monomers (G-actin) polymerize to form arrays of actin filaments (F-actin). F-actin has two structurally and biochemically distinct ends: a barbed end and a pointed end. Polymerization and depolymerization occur at both ends but polymerization is faster at the barbed end. In the absence of actin-binding proteins, F-actin is said to “treadmill” when it reaches steady state; G-actin continuously polymerizes at the barbed end and depolymerizes from the pointed end [3], [4].