p gsk3 β  (Proteintech)

Journal: bioRxiv

Article Title: MAP4-MAP7D1 partitioning on tyrosinated-detyrosinated microtubules coordinates lysosome positioning in nutrient signalling

doi: 10.1101/2025.10.07.680844

Figure Lengend Snippet: MAP7D1 and MAP4 nanoclusters are largely exclusive on microtubule network. (a) Dual-colour confocal images of BS-C-1 cells showing the distribution of microtubule-bound MAP7D1 (magenta) and MAP4 (green). Magenta arrows indicating MAP7D1-dominated microtubules, white arrows indicating MAP4-dominated microtubules, and yellow arrowheads marking microtubules decorated by both MAPs. A corresponding line intensity plot along the yellow line in the zoomed image reveals that the two MAPs are predominantly associated with distinct microtubule tracks. (b) Two-colour STORM image showing the nanoscale distribution of MAP7D1 (magenta) and MAP4 (green) along microtubule tracks. The two MAPs form discrete nanoclusters along microtubule tracks. (c–e) Representative dual-colour STORM images of individual microtubule segments showing examples of MAP7D1-dominant (c), MAP4-dominant (d), and regions with both MAPs (e). (f) Pie-chart depicting the distribution of microtubule segments populated with either MAP4 or MAP7D1 or both. (g) The percentage localization density of each MAP was quantified for individual ROIs and plotted as a stacked column graph. ROIs were sorted in ascending order of MAP4 localization density, revealing an inverse correlation between MAP4 and MAP7D1 localization distribution along microtubules. (h) Representative two-colour STORM images of MAP4 and MAP7D1 nanoclusters classified into four categories based on the extent of spatial overlap. Corresponding histograms show the percentage overlap between MAP4 and MAP7D1 localizations plotted along the x-axis. (i) Pie chart shows the proportion of MAP4–MAP7D1 nanocluster overlap: non-overlapping, small overlap (<20%), partial overlap (20–80%), and high or complete overlap (>80%). This data further supports the largely non-overlapping nature of the two MAPs. Scale bars: 10 μm for (a), 5 μm for zoomed images; 1 μm for (b); 500 nm for (c–e); 100 nm for (g).

Article Snippet: Microtubule Binding Domain (MTBD) of MAP4 mRuby-MAP4-C-10 (Addgene Plasmid #55873 from Michael Davidson).

Techniques:

Journal: bioRxiv

Article Title: MAP4-MAP7D1 partitioning on tyrosinated-detyrosinated microtubules coordinates lysosome positioning in nutrient signalling

doi: 10.1101/2025.10.07.680844

Figure Lengend Snippet: MAP4 preferentially associates with tyrosinated microtubules. Two-color super-resolution SIM images of endogenous MAP4 (magenta) with (a) tyrosinated, (b) detyrosinated, and (c) acetylated microtubules (green) in BS-C-1 cells, with corresponding line intensity plots showing preferential MAP4 localization to tyrosinated microtubules. (d) Three-color SIM image of MAP4 (magenta) with tyrosinated (green) and acetylated (blue) microtubules. Arrowheads indicate MAP4 associated with acetylated-tyrosinated microtubules; arrows indicate MAP4 associated with acetylated-detyrosinated microtubules. (e) Quantification of MAP4 localization across distinct microtubule subsets from n = 29 ROIs per subset from 12 different cells. MAP4 fluorescence intensity was measured from ROIs of equal size drawn on each microtubule population, ensuring uniform sampling across microtubule populations. (f) Percentage of MAP4 associated with each subset was calculated by dividing the mean MAP4 intensity on that subset by the total MAP4 intensity across all ROIs. (g) Two-color STORM images of MAP4 (magenta) with tyrosinated microtubules (green). HDBSCAN-based cluster analysis quantifies (h) inter-cluster distance and (i) cluster density (number of clusters per unit length), revealing significantly higher MAP4 clustering on tyrosinated microtubules (yellow box) compared to detyrosinated microtubules (cyan box). ROIs for detyrosinated microtubules were selected based on MAP4-decorated microtubule patterns lacking tyrosination signal. n = 26 ROIs were analyzed per microtubule subset from a cell. Data represents mean (line) ± SD (box). Statistical significance was assessed using the Mann–Whitney U test (****p < 0.0001). Scale bars: 10 μm for (a–c) and corresponding zoomed images at 5 μm; 5 μm for (d), 2 μm for (g), and corresponding zoomed images at 500 nm.

Article Snippet: Microtubule Binding Domain (MTBD) of MAP4 mRuby-MAP4-C-10 (Addgene Plasmid #55873 from Michael Davidson).

Techniques: Fluorescence, Sampling, MANN-WHITNEY

Journal: bioRxiv

Article Title: MAP4-MAP7D1 partitioning on tyrosinated-detyrosinated microtubules coordinates lysosome positioning in nutrient signalling

doi: 10.1101/2025.10.07.680844

Figure Lengend Snippet: MAP7D1, not MAP4, is sensitive to lattice expansion. (a-d) Three-color confocal images and corresponding line intensity profiles of BS-C-1 cells (a) DMSO-treated cells immunostained for endogenous MAP4 (magenta) with acetylated (green) and tyrosinated (blue) microtubules. Arrowheads indicate MAP4 colocalization with acetylated-tyrosinated microtubules; arrows indicate MAP4 exclusion from acetylated-detyrosinated microtubules. (b) Taxol-induced lattice expansion preserves MAP4’s preference for acetylated-tyrosinated microtubules. (c) DMSO-treated cells immunostained for endogenous MAPD1 (magenta) with acetylated (green) and tyrosinated (blue) microtubules. Arrowheads indicate MAP7D1 sparsely decorated on acetylated-tyrosinated microtubules; arrows indicate MAP7D1 enriched on acetylated-detyrosinated microtubules. (d) Taxol-induced lattice expansion redistributes MAP7D1 to acetylated-detyrosinated (arrows) and acetylated-tyrosinated (arrowheads) microtubules. (e) Super-resolution STORM images showing endogenous MAP4 (magenta) or MAP7D1 (magenta) localization on tyrosinated microtubules in DMSO or Taxol-treated cells. ROIs were drawn on tyrosinated microtubules based on corresponding two-color TIRF images (shown as insets, also see Figure S3b, c). Yellow box shows the corresponding zoomed ROIs, highlighting unaltered MAP4 localization but increased MAP7D1 density on tyrosinated microtubules after lattice expansion. (h) Quantification of MAP4 and MAP7D1 localization density on tyrosinated microtubules from STORM images. No. of ROIs analyzed, MAP4 DMSO ( n =383), MAP4 Taxol (n= 658), MAP7D1 DMSO (n= 317), MAP7D1 Taxol (n= 374) from 3 different cells. Data represents mean (line) ± SD (box). Statistical significance was assessed using the Mann–Whitney U test (****p < 0.0001, not significant [ns]). Scale bars: 10 μm for (a–d), with zoomed images at 2 μm; 2 μm for (e), with zoomed images at 500 nm.

Article Snippet: Microtubule Binding Domain (MTBD) of MAP4 mRuby-MAP4-C-10 (Addgene Plasmid #55873 from Michael Davidson).

Techniques: MANN-WHITNEY

Journal: bioRxiv

Article Title: MAP4-MAP7D1 partitioning on tyrosinated-detyrosinated microtubules coordinates lysosome positioning in nutrient signalling

doi: 10.1101/2025.10.07.680844

Figure Lengend Snippet: MAP4 projection domains restrict its association to tyrosinated microtubules. (a) The Schematic of MAP4 truncation constructs used in the study. (b-e) Representative confocal images of BS-C-1 cells transiently expressing GFP-tagged MAP4 constructs (magenta) immunostained for detyrosinated microtubules (green) along with corresponding insets show magnified views of the regions indicated by yellow boxes. (f) Box plot quantifying MAP4 colocalization with detyrosinated microtubules. (g) Model illustrating the role of MAP4 projection domains in conferring specificity for tyrosinated microtubules. Data represents mean (line) ± SD (box). Number of cells (n) analyzed for MAP4-FL (10), MAP4-MTBD (11), MAP4-ΔN (15) and MAP4-ΔC (14). Statistical significance was assessed using the Mann– Whitney U test (*p<0.05, **p<0.01, not significant [ns]). Scale bars: 10 μm.

Article Snippet: Microtubule Binding Domain (MTBD) of MAP4 mRuby-MAP4-C-10 (Addgene Plasmid #55873 from Michael Davidson).

Techniques: Construct, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: MAP4-MAP7D1 partitioning on tyrosinated-detyrosinated microtubules coordinates lysosome positioning in nutrient signalling

doi: 10.1101/2025.10.07.680844

Figure Lengend Snippet: Kinesin-1 and kinesin-3 rigor mutants differentially associate with MAP4 and MAP7D1 (a-b) Confocal images of BS-C-1 cells transiently expressing GFP-tagged (a) KIF5B-R (green) or (b) KIF1A-R (green), immunostained for endogenous MAP4 (magenta). (c) Bar plot of Mander’s colocalization quantification shows significantly higher colocalization of MAP4 with KIF1A-R ( n = 8 cells) than KIF5B-R ( n = 9 cells). (d-e) Confocal images of cells expressing GFP-tagged (d) KIF1A-R or (e) KIF5B-R (green), immunostained for endogenous MAP7D1 (magenta). (f) Bar plot of Mander’s colocalization quantification reveals significantly higher colocalization of MAP7D1 with KIF5B-R ( n = 10 cells) than KIF1A-R ( n = 9 cells). (g–h) Co-expression of KIF5B-R (green) with (g) MAP4-FL or (h) MAP4-MTBD (magenta), Line intensity profiles show MAP4-FL is excluded from KIF5B-R-decorated microtubules, while MAP4-MTBD exhibits extensive colocalization. (i) Model illustrating the preferential association of KIFB-R and KIF1A-R with MAP7D1 and MAP4, respectively. Bars represent the mean; whiskers indicate standard deviation from three independent experiments. Statistical significance was assessed using the Mann–Whitney U test (****p < 0.0001). Scale bars: 10 μm.

Article Snippet: Microtubule Binding Domain (MTBD) of MAP4 mRuby-MAP4-C-10 (Addgene Plasmid #55873 from Michael Davidson).

Techniques: Expressing, Standard Deviation, MANN-WHITNEY

Journal: bioRxiv

Article Title: MAP4-MAP7D1 partitioning on tyrosinated-detyrosinated microtubules coordinates lysosome positioning in nutrient signalling

doi: 10.1101/2025.10.07.680844

Figure Lengend Snippet: (a) Correlative live-cell and STORM imaging of lysosomes with MAP4 in BS-C-1 cells. Single-particle trajectories (SPT) from live imaging were aligned and overlaid on the corresponding STORM image of MAP4. Anterograde track is shown in yellow and retrograde track in cyan. In corresponding graphs, the trajectories are colour-coded to indicate run phases (blue) versus pause phases (red), revealing more processive retrograde runs than anterograde runs. (b) Scatter plot of lysosome speed versus MAP4 density. Dotted lines mark the mean on both axes. Points below (pink) or above (blue) the mean MAP4 density illustrate that lysosomes show reduced average speed at higher MAP4 density. (c) Paired correlation plot between lysosomal speed and MAP4 density, separated into anterograde (n = 13 trajectories) and retrograde (n = 11 trajectories) tracks. This shows that anterograde-moving lysosomes experience greater hindrance in speed with increasing MAP4 density. (d) Paired correlation plot between run-phase and MAP4 density for the same anterograde (n = 13) and retrograde (n = 11) tracks. Higher MAP4 density reduces run-phase more markedly for anterograde than for retrograde tracks. (e) Correlative live-cell and STORM imaging of lysosomes with MAP7D1 (magenta) in BS-C-1 cells. Single-particle trajectories of lysosomes from live imaging were aligned and overlaid on the STORM image of MAP7D1. Anterograde trajectories are marked in yellow and retrograde in cyan. Run phases (blue) and pause phases (red) highlight that retrograde runs are more processive than anterograde runs. (f) Scatter plot of lysosome speed versus MAP7D1 density. Dotted lines mark the mean on both axes. Points below (pink) or above (blue) the mean MAP7D1 density indicate that lysosomes have reduced average speed at higher MAP7D1 density. (g) Paired correlation plot between lysosomal speed and MAP7D1 density, separated into anterograde (n = 14 trajectories) and retrograde (n = 17 trajectories) tracks. Anterograde-moving lysosomes show greater hindrance in speed as MAP7D1 density increases. (h) Paired correlation plot between run-phase and MAP7D1 density for the same anterograde (n = 14) and retrograde (n = 17) tracks. Higher MAP7D1 density reduces run-phase measures more for anterograde than retrograde tracks. Scale bars: 500 nm.

Article Snippet: Microtubule Binding Domain (MTBD) of MAP4 mRuby-MAP4-C-10 (Addgene Plasmid #55873 from Michael Davidson).

Techniques: Imaging, Single Particle

Journal: bioRxiv

Article Title: MAP4-MAP7D1 partitioning on tyrosinated-detyrosinated microtubules coordinates lysosome positioning in nutrient signalling

doi: 10.1101/2025.10.07.680844

Figure Lengend Snippet: MAP density on microtubules regulates nutrient-dependent lysosomal positioning (a) Representative confocal images of BS-C-1 cells immunostained for endogenous LAMP2 (green) showing lysosomal distribution and (b) radial intensity profile plot for quantification of lysosomal distribution under control (n=27 cells), nutrient starvation (n=17 cells) and 2x amino acids stimulated (n=17 cells) conditions, (c-f) Representative STORM images MAP4 and MAP7D1 under control, nutrient starvation and nutrient stimulation along with corresponding localization density distribution plots from 3 different cells in each conditions. (g-h) Representative confocal images of control cells showing lysosomal distribution along with the corresponding radial intensity profile plot in endogenous (n=27 cells), MAP4-FL overexpression (n=27 cells) and MAP7-FL overexpression (n=23 cells) conditions. (i-j) Representative confocal images of untreated control cells showing lysosomal distribution along with corresponding radial intensity profile plot in scramble siRNA (n=21 cells), MAP4 siRNA (n=37 cells) and MAP7D1 siRNA (n=27 cells). (k-l) Representative confocal images of nutrient-stimulated cells showing lysosomal distribution along with corresponding radial intensity profile plot in scramble siRNA (n=23 cells), MAP4 siRNA (n=41 cells) and MAP7D1 siRNA (n=31 cells). Lysosomal distribution as a function of distance from the center of the nucleus was quantified from confocal images using the Radial Profile plugin in ImageJ (see Methods for details). Radial intensity profile graphs represent the mean ± SEM of lysosomal intensity profiles, averaged from three independent experiments. Statistical significance was assessed using the Kolmogorov–Smirnov (KS) test, comparing treated conditions to the control (n.s.> 0.05, *** < 0.05, **** p < 0.00001). Scale bars: 10 μm for confocal images, 5 μm for STORM images in c, e and corresponding insets at 1 μm.

Article Snippet: Microtubule Binding Domain (MTBD) of MAP4 mRuby-MAP4-C-10 (Addgene Plasmid #55873 from Michael Davidson).

Techniques: Control, Over Expression

Journal: bioRxiv

Article Title: MAP4-MAP7D1 partitioning on tyrosinated-detyrosinated microtubules coordinates lysosome positioning in nutrient signalling

doi: 10.1101/2025.10.07.680844

Figure Lengend Snippet: Proposed model The schematic presents a model integrating findings from this study and prior reports , , , . Under homeostatic conditions, the microtubule network is differentially decorated with MAPs: MAP4 is enriched on tyrosinated microtubules, while MAP7D1 is enriched on detyrosinated microtubules. These MAP–PTM combinations establish specialized tracks that spatiotemporally regulate lysosome transport. On MAP7D1-decorated detyrosinated microtubules, kinesin-1 is preferentially recruited but its motility is dampened by the high density of MAP7D1 nanoclusters, promoting lysosomal immobilization in the perinuclear region to support autophagy-related functions. In contrast, MAP4-enriched tyrosinated microtubules favor fast kinesin-3 or kinesin-2–driven anterograde transport, redistributing lysosomes toward the cell periphery to enhance mTORC1 signaling. Importantly, anterograde transport is dynamically balanced by dynein-driven retrograde movement, which counteracts kinesin activity and maintains equilibrium between perinuclear and peripheral lysosome pools during homeostasis. Upon nutrient modulation, MAP densities are remodelled along microtubule tracks. Nutrient deprivation increases MAP7D1 density and reduces MAP4 abundance, thereby immobilizing kinesin-1 and biasing transport toward dynein-driven retrograde motility, which retains lysosomes in the perinuclear region. Conversely, nutrient stimulation decreases MAP7D1 density, while maintaining optimal MAP4 density, thereby enabling faster motility of kinesin and promoting anterograde transport and peripheral positioning of lysosomes. This adaptive tuning of MAP organization in response to nutrient availability provides a mechanism for spatial reorganization of lysosomes to meet cellular metabolic demands.

Article Snippet: Microtubule Binding Domain (MTBD) of MAP4 mRuby-MAP4-C-10 (Addgene Plasmid #55873 from Michael Davidson).

Techniques: Activity Assay