Fig 2: MAP7 enhances kinesin-1 binding to microtubules in COS cells.(A) Confocal images of live COS cells expressing KIF5C(1-560)-mNeGr (green) with mCherry or MAP7-FL-mCherry (red). High magnification images are shown in the bottom panel of each condition. (B, C) Line scan analysis (B) of the KIF5C(1-560)-mNeGr fluorescence intensity from the dashed lines in (A), and comparison of the peak/background signals from the mNeGr fluorescence in cells expressing mCherry or MAP7-FL-mCherry (C). ***p=0.0004 from t-test (Mean ±SEM). n = 5 lines from one cell. (D) Confocal images of a region in a live COS cell triple transfected to express MAP7-FL-iRFP (red), KIF5C(1-560)-mNeGr (green), and tubulin-mCherry (white). (E) High magnification confocal images of COS cells expressing KIF5C(1-560)-mNeGr (green) with mCherry fusion proteins (red) of the indicated MAP7 fragments.10.7554/eLife.36374.015Figure 5—source data 1.Line scan data and quantification of peak/background ratio in Figure 5B,C.

Fig 3: Distinct roles of MAP7 domains in DRG axon growth and branching.(A) Primary structure of MAP7, indicating the phosphorylation (P) domain and the two coiled-coil (CC) regions that interact with microtubules (MT(CC1)) and kinesin-1 (Kinesin(CC2)). The full length (FL) MAP7 and various fragments used in the study are illustrated by line drawings. (B) Representative images of neurofilament staining in E14 rat DRG neurons expressing EGFP or EGFP-tagged fusion proteins of MAP7-FL or various MAP7 fragments. Arrows point to interstitial branches. (C) Quantification of the number of branches per cell as measured by counting the total number of tips per neuron in E14 DRG neurons expressing EGFP or EGFP fusion proteins. Branches were further divided into two groups: terminal branches arising from the distal 10% part of the axon and interstitial branches arising from the rest of the axons. n = 33, 26, 46, 39, 20, 51, 31, 14 for EGFP, FL, ΔC, ΔN, ΔP, N, P and C respectively. ANOVA-test (Mean ±SEM): EGFP-FL, p=0.013; EGFP-ΔC, p≤0.0001; EGFP-ΔN, p=0.98. (D) Quantification of the total length of main axons in neurons expressing different MAP7 constructs. n = 44, 21, 18, 22, 21, 77, 12, 15 for EGFP, FL, ΔC, ΔN, ΔP, N, P and C respectively. ANOVA-test (Mean ±SEM): EGFP-FL, p=0.0003; EGFP-ΔC, p=0.29; EGFP-ΔN, p≤0.0001. (E) Comparison of the branch length between MAP7-FL-EGFP and MAP7-ΔC-EGFP expressing DRG neurons. n = 36 for FL and 73 for ΔC. T-test (Mean ±SEM): p=0.04. *p<0.05; **p<0.01; ***p<0.001; ns: not significant. Scale bar: 200 µm.10.7554/eLife.36374.004Figure 1—source data 1.Data for the measurement of branch number, axon length, and branch length in Figure 1C–E.

Fig 4: Analysis of kinesin-1 domains for its recruitment to MAP7-bound microtubules.(A) Domain structure of KIF5C, which includes motor head, stalk1, and stalk2 domains. The structures of various truncated constructs used in this study are illustrated by line drawings. (B) Confocal images of live COS cells expressing MAP7-FL-mCherry (red) with mNeGr fusions of the indicated KIF5C domains. (C) Sequential confocal images of KIF5C(1-560)-mNeGr (green) and MAP7-FL-iRFP (red) in live COS cells before (pre) and after photobleaching at various time points. (D) FRAP recovery plots for the mNeGr fusion proteins of the active motor KIF5C(1-560) and the stalk1 domain KIF5C(335-560), as well as MAP7-FL-iRFP. Solid lines are derived from non-linear curve fitting. (E, F) Comparison of the off-rate constant (koff) and the total recovery derived from curve fitting for the mNeGr fusion proteins of KIF5C(1-560) and KIF5C(335-560) on MAP7-FL-iRFP-bound microtubules. n = 182 for KIF5C(1-560) and 238 for KIF5C(335-560) from seven cells each condition. T-test (Mean ±SEM) for koff (E): p=0.03 and for recovery (F): p=0.65. *p<0.05; and ns: not significant. Scale bars: 2 μm.10.7554/eLife.36374.017Figure 6—source data 1.Data for the fluoresence recovery curve (D) and the kinetic parameters (E,F) in Figure 6.

Fig 5: FRAP analysis of MAP7 binding to microtubules in COS cells.(A) Representative sequential confocal images of N-EGFP or FL-EGFP on single microtubules in live COS cells before (pre) and after photobleaching at various time points. (B) Fluorescence recovery plots for N-, P-, and FL-EGFP fusion proteins along single microtubules in COS cells. Solid lines are derived from non-linear curve fitting based on the inverse of an exponential decay model. (C, D) Comparison of the dissociation rate constant (koff) (C) and the total recovery (D) derived from curve fitting of the MAP7 N-, P-, FL-, and ΔC-EGFP fluorescence recovery. n = 321 for N (11 cells), 396 for P (9 cells), 420 for FL (7 cells), and 150 for ΔC (5 cells). ANOVA-test (Mean ±SEM) for koff (C): N-P, p=0.001; P-FL, p=0.99; FL-ΔC, p=0.99, and for recovery (D): N-P, p=0.39; P-FL, p=0.001; FL-ΔC, p=0.018. *p<0.05; ***p<0.001; ns: not significant. Scale bar: 2 μm.10.7554/eLife.36374.013Figure 4—source data 1.Data for the fluoresence recovery curve (Figure 4B) and the kinetic parameters (Figure 4C,D).