Ectopic tethering of KNL-1 to the PVD plasma membrane results in the formation of actin clusters. (A) Top: Cartoon of the approach employed for the ectopic tethering of KNL-1 to the plasma membrane of the PVD neuron using a myristoylation domain (MYR). Bottom: Schematic illustrating the functional domains within KNL-1. (B) Images showing the morphology of the PVD cell body and proximal primary dendrite at late L2 larvae stage (green) and actin (magenta) in MYR::TagBFP (MYR), KNL-1::TagBFP (KNL-1), and MYR::KNL-1::TagBFP (MYR::KNL-1). Actin is visualized using Lifeact::mKate2, and the plasma membrane is labeled with mNeonGreen(mNG)::PH. The white arrowheads indicate the actin clusters. Scale bar, 5 µm. (C) Quantification of the number of actin clusters in the PVD dendrites in the indicated conditions. n represents the number of animals. Error bars represent mean ± 95% CI. P values from Kruskal–Wallis test followed by Dunn’s test. (D) Time-lapse imaging of actin cluster membrane dynamics in MYR::KNL-1 animals (top) Scale bar, 2 µm. Kymograph of mNG::PH at the membrane protrusion indicated by the dashed selection (bottom). Scale bar, 1 µm. and 5 min, respectively. (E) Images showing the morphology of PVD membrane at late L2 larvae stage (green) and actin (magenta) in animals expressing MYR:: KNL-1^1–505 or MYR::KNL-1^505–1010. Scale bar, 5 µm. (F) Quantification of the number of actin clusters in the PVD dendrites in indicated conditions. n represents number of animals. Error bars represent mean ± 95% CI. P values from Kruskal–Wallis test followed by Dunn’s test. (G) Images showing morphology of PVD membrane at late L2 larvae stage (green) and actin (magenta) in animals expressing MYR::KNL-1^MELA and MYR::KNL-1^SAAA+RRASA. Scale bar, 5 µm. (H) Quantification of the number of actin clusters in the PVD dendrites in indicated conditions. n represents number of animals. Error bars represent mean ± 95% CI. P values from Kruskal–Wallis test followed by Dunn’s test.