For DAAM1, the recovery half time was approximately 3 ~ 8 fold slower than GFP. GUID:?F8CA5DD6-579E-45BB-8FAB-7D3F27A44987 S3 Fig: Testing and tracking of photoactivated molecules. (A) An example of simultaneous FRAP of GFP–actin and photoactivation of PATagRFP-DAAM1. Left image is the colored maximum intensity projection (MIP) of 1 1 sec post-activation. Right image is usually GFP–actin one frame before activation/bleaching. The FRAP/activation region (ROI) is usually highlighted in reddish. A kymograph of FRAP recovery is usually shown for the ROI region. (B) PATagRFP decays drastically over a short period of time. Measurements were taken using relatively immobile PATagRFP-Filamin A. Single spot activation with 405 nm laser was defined using round ROI with 15-pixel diameter. Acquisition using 561 nm excitation between 40 mW?cm-2 to 80 mW?cm-2 with various exposure occasions reduced the intensity to background level within 10 sec. (C) and (D) closer look of the PATagRFP-DAAM1 dynamics. A region from a non-treated MEF cell transfected with GFP–actin and PATagRFP-DAAM1 R306465 was enlarged. The 1 sec MIP of PATagRFP-DAAM1 was merged with the GFP–actin. The DAAM1 spots localized on stress fiber were highlighted using R306465 reddish circles. The movements of these PATagRFP-DAAM1 spots were tracked as shown in (D). (E) With longer interval between each acquisition time, PATagRFP-DAAM1 spots were found without movement. Most of these spots, highlighted in reddish circles, colocalized with actin nodes.(TIFF) pone.0163915.s003.tiff (17M) GUID:?FC5B33D2-6D9D-496B-B390-F9460B3090CB S1 Movie: Actin structure switch during the treatment of Latrunculin A and wash off. HeLa JW cells were transfected with Ruby-Lifeact and GFP-DAAM1 24 hours before imaging. 500 nM LatA was first added to the cells. Wash off was carried out by replacing the LatA-containing medium to normal DMEM after 3 times washing done within 3 minutes. The movie showed the Ruby-Lifeact (magenta in merged image), GFP-DAAM1 (green in merged image) channels and the merged of the two channels. Rolling ball background subtraction was carried Col4a3 out to GFP channel to increase the contrast. The lower right region of the cell was enlarged to show the details. The first few minutes after LatA wash off was slowed down 4 times in the enlarged region. S1 Movie corresponds to Fig 2AC2E.(AVI) pone.0163915.s004.avi (94M) GUID:?19C0A2DE-E879-43C6-A6EB-7BAC6C5FE64F S2 Movie: DAAM1-NC mutant is an R306465 active formin. H460 cells were transfected with GFP-DAAM1-NC and Ruby-Lifeact (left) or GFP-DAAM1-NCI698A and Ruby-Lifeact (right). The movie starts at 17 moments of LatA treatment and continues for 9 moments. S2 Movie corresponds to Fig 3D.(AVI) pone.0163915.s005.avi (15M) GUID:?CB0A05E1-60E6-4C53-BCC7-2625171277CA S3 Movie: Actin nodes are transient in rapamycin induced DAAM1 membrane translocation system. HeLa JW cells were transfected with Lyn11-FRB, CFP-FKBP, GFP-FKBP-DAAM1, Ruby-Lifeact and treated with 1 uM Rapamycin and 500 nM LatA. The movie starts at the beginning of LatA treatment and lasts for 40 minutes. Frames with actin nodes appearance are indicated in the movie. S3 Movie corresponds to Fig 5A.(AVI) pone.0163915.s006.avi (63M) GUID:?9D31DE87-098E-4777-B114-CA18BA855DF3 Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract A nodal cytoplasmic actin network underlies actin cytoplasm cohesion in the absence of stress fibers. We previously described such a network that forms upon Latrunculin A (LatA) treatment, in which formin DAAM1 was localized at these nodes. Knock down of DAAM1 reduced the mobility of actin nodes but the nodes remained. Here we have investigated DAAM1 containing nodes after LatA washout. DAAM1 was found to be distributed between the cytoplasm and the plasma membrane. The membrane binding likely occurs through an interaction with lipid rafts, but is not required for F-actin assembly. Interesting the R306465 forced interaction of DAAM1 with plasma membrane through a rapamycin-dependent linkage, enhanced F-actin assembly at the cell membrane (compared to the cytoplasm) after the LatA washout. However, immediately after addition of both rapamycin and LatA,.