Fig 1.
Long-term FRAP experiment is demonstrated to explain the importance of considering the effect of intracellular advection, where the intensity recovery in the fixed region of interest displays an abnormal curve.
(A) The rectangular region in the left image (mClover2-tagged β-actin in an A7r5 cell) is magnified on the right panels, in which FRAP responses are shown. The arrow heads represent the bleached region that is being spatially shifted over time; red, green, and blue represent an identical part of the bleached rectangular region at t = 0 400, and 800 s after the photobleaching, respectively. (B) Time-series change in fluorescence intensity obtained based on a conventional approach using a spatially fixed region of analysis. The FRAP response is not properly approximated by the typical approach using exponential curves. (C) The intensity profiles along the length of a single SF in a spatially fixed frame at t = 0, 400, and 800 s. Scale, 10 μm.
Fig 2.
Schematic of the equilibrium distribution and initial condition in the numerical model.
(A) The equilibrium distribution (green) corresponds to the intensity distribution of a single SF. Purple arrows represent the velocity vectors. (B) The initial condition is expressed by the combination of post-bleach intensity distribution (yellow) and the equilibrium distribution (green).
Table 1.
Parameters used for the numerical analysis in Fig 3.
Fig 3.
Numerical simulation validates the advection-reaction model.
(A) The pre-bleach distribution expressed by Eq (8). The arrows represent the velocity vectors. (B) FRAP responses simulated by the first-order upwind scheme (see S1 Video). (c) FRAP responses fitted by the least-square method (see S2 Video).
Table 2.
Characteristic parameter α used for the numerical analysis and determined by the least-square method in Fig 3.
Fig 4.
The advection-reaction model fitted to experimental data.
(A) FRAP was performed onto SFs in an A7r5 cell, in which mClover2-tagged β-actin is expressed. (B) Experimentally obtained two-dimensional FRAP response of a single SF (rectangular region in A; see S3 Video). (C) The model was fitted with the least-square method to the spatiotemporal evolution of the experimentally obtained FRAP response (see S4 Video). The arrows represent the velocity vectors. Scale, 10 μm. (D) The coefficient of determination in the regression analysis by using the advection-reaction model with or without the velocity shown by mean ± SD (n = 66 for each), in which the former case determines all parameters θ = (K0, koff, Ux, Uy), whereas the latter case does θ = (K0, koff, 0, 0).
Fig 5.
The effect of actin–myosin interaction and actin polymerization on the inherent properties of actin on SFs and intracellular advection.
(A–C) Cells expressing mClover2-tagged β-actin and undergoing FRAP at vehicle control (A), Blebb (B), and LatA (C) conditions. Kymographs of the fluorescence intensity along the specified yellow lines are shown below for each condition. (D), (E) The dissociation rate (D) and advection velocity (E) of actin on SFs for each condition shown by mean ± SD (n = 66, 24, and 52 regions of interest for vehicle control, Blebb, and LatA conditions, respectively, from at least 3 independent experiments). Box and whisker plots show the following: The upper and lower edges of the boxes represent the 75 and 25 percentile ranges, respectively; the central lines represent the median; the whiskers represent the standard deviation; the open dots represent the mean; and, the closed dots represent outliers. The asterisks indicate significant differences. (F)–(H) The histogram of the orientation index that describes the correlation in the directions of SFs and advection velocity vectors for vehicle control (F), Blebb (G), and LatA (H) conditions (n = 66, 24, and 52 regions of interest for vehicle control, Blebb, and LatA conditions, respectively, from at least 3 independent experiments). Scale, 10 μm.
Fig 6.
Schematic to illustrate the actin turnover in the sarcomeric unit of SFs at the different drug treatment conditions.
With Blebb treatment, the entire actin filaments are dissociated from the sarcomeric unit to increase the actin dissociation rate. Meanwhile, the unbundled and thus movable actin filaments may now contribute to the increase in the advection velocity (yellow arrows). With LatA treatment, the resulting increase in actin monomers in the cytoplasm may slow the depolymerization of preexisting actin filaments and thus decrease the actin dissociation rate. Meanwhile, the sarcomeric unit tends to slide quickly (yellow arrows) potentially because of the maintained NMII activity, partly damaged physical interface at the adhesions, and activated lamellipodia/filopodia-driven actin motility.