LIM and SH3 protein 1 localizes to the leading edge of protruding lamellipodia and regulates axon development

FIGURE 1:. LASP1 is expressed in the developing brain and localizes to the axon growth cone. (A) Top, a representative Western blot showing the developmental profile of LASP1 in rat hippocampi at the indicated stages. Bottom, the plot shows normalized LASP1 levels relative to tubulin loading control from three independent blots. (B) Representative confocal images of a rat hippocampal neuron in culture at 2 d in vitro (DIV2) stained for endogenous LASP1 (magenta), F-actin (phalloidin, cyan), and α-tubulin (yellow). Individual channels of the growth cone are shown as the insets on the right. Arrows indicate the minor processes destined to be dendrites, and asterisk indicates the axonal growth cone. Scale bar: 20 µm. (C) Representative confocal images of growth cones from fixed DIV2 cultured rat hippocampal neurons, stained for endogenous LASP1 (magenta) and F-actin (phalloidin, cyan). LASP1 localizes to the leading edge of lamellipodia (orange arrowheads) and actin bundles in filopodia (white arrowheads). Scale bars: 5 µm. (D) Representative confocal images of two CAD cells labeled for LASP1 (magenta) and F-actin (phalloidin, cyan). LASP1 is seen at lamellipodia and filopodia in a similar…

FIGURE 2:. GFP-LASP1 localizes to protruding membranes. (A) Representative images of live CAD cells coexpressing intact (CMVΔ0 GFP-LASP1, top) or crippled (CMVΔ4 GFP-LASP1, bottom) CMV promoter-driven GFP-LASP1 (left, magenta), along with Lifeact-mRuby (F-actin, middle, cyan). GFP-LASP1 overexpressed by an intact CMV promoter localizes to the leading edge (orange arrowheads) and actin bundles (white arrowheads), whereas CMVΔ4 GFP-LASP localizes only to the leading edge, similar to endogenous LASP1. Scale bars: 5 µm. (B) Top, representative anti-LASP1 and anti-tubulin Western blots from CAD cells transfected with CMVΔ0 GFP-LASP1 or CMVΔ4 GFP-LASP1, along with nontransfected controls (NTF). Bottom, graph shows quantification of GFP-LASP1 expression, normalized to tubulin loading control, with endogenous LASP1 from the nontransfected condition set to 1. Error bars represent standard error. (C) Time-lapse images of CAD cells coexpressing CMVΔ4 GFP-LASP1 and Lifeact-mRuby. Bottom row, images show GFP to mRuby ratio, color coded with a rainbow heat map (scale to right). Images were captured every 5 s for 20 min. Scale bar: 10 µm. Right, representative kymographs (correspond to labeled arrows on…

FIGURE 3:. The LIM domain and nebulin repeats are essential for LASP1 localization to the leading edge. (A) Live CAD cells coexpressing Lifeact-mRuby (F-actin, cyan) and wild-type GFP-LASP1, GFP-LASP1ΔLIM, GFP-LASP1ΔNebulin (GFP-LASP1ΔNeb), or GFP-LASP1ΔSH3 (magenta). Wild-type GFP-LASP1 and GFP-LASP1ΔSH3 localize to the leading edge. However, GFP-LASP1ΔLIM and GFP-LASP1ΔNeb do not localize to the leading edge and instead appear mostly soluble. Scale bars: 10 µm. (B) Line scan analysis of GFP-LASP1 localization at the leading edge (120 line scans from 30 cells across three independent culture replicates). Error bars represent standard error.

FIGURE 4:. LASP1 localizes to actin plus ends at the leading edges of CAD cells. (A) Representative images of CAD cells expressing soluble GFP or CMVΔ4 GFP-LASP1 (magenta), with actin plus ends labeled with rhodamine–conjugated actin (cyan). Scale bars: 5 µm. Graphs (right) depict the average of four normalized line profiles from cells on left (arrows in merged images show location of line scans). Error bars represent standard error. LASP1 localization at the leading edge overlaps with the labeled plus ends. Representative of 10 cells from three independent culture replicates. (B) Representative images showing colocalization of CMVΔ4 GFP-LASP1 (magenta) and endogenous CP in CAD cells (cyan). Scale bar: 5 µm. Graph (right) shows the average of four normalized line profiles from arrows in merged image. Error bars represent standard error.

FIGURE 5:. LASP1 localization to the leading edge depends on barbed ends of actin filaments. (A–D) Time-lapse images of CAD cells expressing CMVΔ4 GFP-LASP1 and Lifeact-mRuby before (time 0), at 5 and 10 min after the application of 25 nM CytoD (A), 100 nM Lat A (B), 10 µM SMIFH2 (C), and 100 µM CK-666 (D). For each condition, representative images of GFP-LASP1, Lifeact-mRuby, and their ratiometric images are shown, except (D) in which only the ratiometric images are shown. For each cell, four kymographs from the lines indicated in the corresponding ratiometric image are shown in the lower portion of each panel. Each kymograph includes the time-lapse recordings of 5 min before (pre-) and 10 min after the onset of the specific drug treatment. Scale bars: 10 µm. Images are representative of three cells per condition across three independent culture replicates. (E) Quantification of LASP1 localization at the leading edge of the cell 5 min before, as well as 1, 5, and 10 min after drug application. Error bars represent standard error. **p < 0.005; ***p < 0.001. P values were calculated using a one-way ANOVA and Holm-Sidak’s multiple comparisons test.

FIGURE 6:. Knockdown of LASP1 truncates axon elongation and reduces branching in cultured hippocampal neurons. (A) Representative anti-LASP1 and anti-tubulin Western blots of lysates from hippocampal neurons transfected with empty plasmid (vector), shLASP1a, or shLASP1b for 72 h. Graph shows ∼60–70% reduction in LASP1 expression in cultures expressing shRNAs across three independent culture replicates. LASP1 levels are normalized to tubulin loading controls. Error bars represent standard error. (B) Axon tracings of representative rat hippocampal neurons transfected at DIV2 with empty backbone control (Vector), shLASP1a, or shLASP1b. Neurons were imaged every 24 h from DIV3 until DIV7, and each day’s growth is temporally color coded (scale lower right, Vector image). Knockdown of LASP1 reduces axon outgrowth over each 24 h period. Scale bar: 150 µm. (C) Graph depicting axon outgrowth (in µm) every 24 h. (D) Graph depicting the number of newly formed branches at each time point. Error bars represent standard error. *p < 0.05; **p < 0.005; ***p < 0.001. P values calculated using a one-way ANOVA Tukey’s HSD post-hoc test. Data collected from approximately 30 neurons from three independent…

FIGURE 7:. Knockdown of LASP1 reduces growth cone protrusion and branch formation in cultured hippocampal neurons. (A) Montages of representative growth cones from DIV5 rat hippocampal neurons expressing Vector control, shLASP1a, or shLASP1b. Montage slices represent 40-min intervals. Scale bar: 40 µm. (B) Graph of growth cone speed (left) and persistence (right). Persistence index represents a ratio of growth cone displacement to total distance traveled. Growth cone speed and persistence are reduced in the absence of LASP1. (C) Graphs of dynamic axon branch formation/elimination (left), and lifetime (right) across all three conditions. Loss of LASP1 causes reduced branch formation and elimination, with no significant effect on the lifetime of new branches. Error bars represent standard error. *p < 0.05; **p < 0.005; ***p < 0.001 by one way ANOVA Tukey HSD post-hoc test. Experiments represent three independent culture replicates. (See Supplemental Tables 3 and 4 for exact n and p values).

FIGURE 8:. Lasp knockdown leads to defects in axon commissure formation in Drosophila. (A) Representative images of stage 16 Drosophila embryos labeled with tau:GFP in egl-expressing ventral nerve cord axons. Top row shows dicer-only control embryo (WT-LASP); bottom row shows embryo expressing dicer plus LASP RNAi (LASP KD). Left column shows GFP-expressing egl neurons (green), middle column shows CNS axon (BP102, magenta), right column shows merged images. Arrowheads indicate defects in commissural axon development, including defasciculation (blue) and axon tracts that do not reach their targets (yellow). Scale bars: 15 µm. (B) Schematic of one segment from wild-type (left) or LASP knockdown (right) egl neurons in the Drosophila ventral nerve cord. Lasp knockdown results in numerous guidance defects, including defasciculation of axon bundles and truncated commissures. (C) Graph shows axon guidance defects from 10 control and 18 knockdown embryos collected across three independent experiments. ***p < 0.001; both defasciculation measures were analyzed using a Student’s t test. Incomplete commissure formation calculated using a Welch’s t test. Error bars represent standard error.