Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Lowery LA
,
Stout A
,
Faris AE
,
Ding L
,
Baird MA
,
Davidson MW
,
Danuser G
,
Van Vactor D
.
Abstract
BACKGROUND: Microtubule (MT) regulators play essential roles in multiple aspects of neural development. In vitro reconstitution assays have established that the XMAP215/Dis1/TOG family of MT regulators function as MT 'plus-end-tracking proteins' (+TIPs) that act as processive polymerases to drive MT growth in all eukaryotes, but few studies have examined their functions in vivo. In this study, we use quantitative analysis of high-resolution live imaging to examine the function of XMAP215 in embryonic Xenopus laevis neurons.
RESULTS: Here, we show that XMAP215 is required for persistent axon outgrowth in vivo and ex vivo by preventing actomyosin-mediated axon retraction. Moreover, we discover that the effect of XMAP215 function on MT behavior depends on cell type and context. While partial knockdown leads to slower MT plus-end velocities in most cell types, it results in a surprising increase in MT plus-end velocities selective to growth cones. We investigate this further by using MT speckle microscopy to determine that differences in overall MT translocation are a major contributor of the velocity change within the growth cone. We also find that growth cone MT trajectories in the XMAP215 knockdown (KD) lack the constrained co-linearity that normally results from MT-F-actin interactions.
CONCLUSIONS: Collectively, our findings reveal unexpected functions for XMAP215 in axon outgrowth and growth cone MT dynamics. Not only does XMAP215 balance actomyosin-mediated axon retraction, but it also affects growth cone MT translocation rates and MT trajectory colinearity, all of which depend on regulated linkages to F-actin. Thus, our analysis suggests that XMAP215 functions as more than a simple MT polymerase, and that in both axon and growth cone, XMAP215 contributes to the coupling between MTs and F-actin. This indicates that the function and regulation of XMAP215 may be significantly more complicated than previously appreciated, and points to the importance of future investigations of XMAP215 function during MT and F-actin interactions.
Figure 1. Knocking down XMAP215 leads to reduced axon outgrowth due to increased rates of axon retraction. (A) Western blot showing knockdown of XMAP215 with increasing levels of morpholino oligonucleotide (MO). (B-C) Confocal images of whole-mount embryo immunostaining for acetylated tubulin shows peripheral axon outgrowth at two days post-fertilization. (D-E) Quantification of axon outgrowth in culture after XMAP215 knockdown (KD). n = explant number (D), axon number (E). (F-I) Phase contrast images of axons from control or XMAP215 KD neural explants (*) after 24 hours of culturing. (J-K) Timelapse montage of representative axons. (See Additional file 1). (L-O) Quantification of axon outgrowth parameters from timelapse imaging, between 18 and 23 hours of culturing. Box-and-whisker plots indicate the mean (diamond), median, extrema and quartiles. *P < 0.05, **P < 0.01, ***P < 0.001 comparing KD with control. ns not significant. n = axon number. Bar is 50 μm for (B,C), 20 μm for (F-K).
Figure 2. Reducing XMAP215 function leads to an increase in microtubule (MT) plus-end velocity in growth cones. (A-F) Data and quantitative analysis of the mean values per growth cone of EB1-GFP comets in control and XMAP215 knockdown (KD) (see Additional file 2). (G, H) Quantification of MT growth track parameters after adding XMAP215 mRNA to KD. (I-J) Quantification of MT plus-end growth velocities in neural crest and axons. Box-and-whisker plots indicate the mean (diamond), median, extrema and quartiles. *P < 0.05, **P < 0.01, ***P < 0.001, ns not significant. N = growth cone/cell number. Bar is 5 μm.
Figure 3. GFP-TOG primarily localizes to microtubule (MT) lattice yet rescues MT plus-end velocity defect of XMAP215. (A) Micrograph of XMAP215-GFP in neuron, with arrowhead pointing to plus-end tracking and arrows to lattice-binding. (See Additional file 3A) (B) Micrograph of GFP-TOG in neuron, with arrow pointing to lattice-binding. (See Additional file 3B) (C) Timelapse montage of representative MT, with mKate2-EB1 (top) and GFP-TOG (bottom). (See Additional file 3C) (D) Quantification of GFP-TOG localization in growth cones and neural crest. n = number MTs examined. (E-G) Quantification of MT parameters. Box-and-whisker plots indicate the mean (diamond), median, extrema and quartiles. *P < 0.05 comparing conditions. ns not significant. n = axonal growth cone number. Bar is 10 μm for (A-B), 1 μm for (C).
Figure 4. XMAP215 knockdown (KD) leads to changes in microtubule (MT) lattice and F-actin flow rates. (A-C) Micrograph of mKate2-tubulin at low levels in control growth cone (A), overlaid with flow vectors calculated by QFSM software at three different time points (B), and (C) rose plot of MT flow directions within all control growth cones, thresholded to include only vectors above the mean, in control. Zero degrees denotes flowing in the retrograde direction, towards the axon, while 180 degrees corresponds to the anterograde direction, with MTs flowing outwards in the direction of new growth (direction of arrow). (See Additional file 4A) (D-F) The same type of data as in (A-C), but for XMAP215 KD growth cones. (See Additional file 4B) (G-J) Micrograph of F-actin labeled by fluorescent kabC in control growth cone (G) and XMAP215 KD (I), with overlay of F-actin flow vectors from QFSM software at three different time points (H,J). (See Additional file 4C,D) (K) Quantification of F-actin speckles using QFSM software. Note that wild-type flow rates are slower than traditional kymograph-measured F-actin retrograde flow rates because manual analysis tends to selectively measure prominent, fast-flowing speckles, while QFSM measures every F-actin speckle within the entire growth cone. For analysis of F-actin flow quantification, only the top quartile of flow vectors for each movie was utilized. This excludes any false positive speckle detection from analysis, and also enhances the likelihood of identifying differences in maximum rates of F-actin retrograde flows. (L-N) Micrograph overlays of EB1-GFP tracks from a two-minute timelapse image series in control (L) and XMAP215 KD (M). (N) Quantification of the percentages of growth cones with co-linear versus non-co-linear tracks. Box-and-whisker plots indicate the mean (diamond), median, extrema and quartiles for each group. ns not significant. n = number of growth cones examined. Scale bar is 5 μm.
Figure 5. Proposed model for XMAP215 function in neurons. (A) Cartoon schematic of proposed functions of XMAP215 in wild-type conditions. Our data suggests that XMAP215 functions to mediate MT-F-actin coupling in both axons and growth cones. In the axon, as XMAP215 knockdown (KD) leads to a myosin II-dependent increase in axon retraction, this implicates XMAP215 as part of the machinery that stabilizes MT forces to oppose the actomyosin-mediated retraction (1). In the growth cone, XMAP215 KD leads to an increase in anterograde MT sliding (2) and a loss of colinearity of MT trajectories (3). As MT translocation rates and trajectory coherence within growth cones are strongly affected by coupling to F-actin retrograde flow, this data suggests that XMAP215 also contributes to the linkages between MTs and F-actin in the growth cone. These novel functions that we propose are in addition to the canonical function of XMAP215 - driving processive MT polymerization (4). (B) Consequences of XMAP215 KD. When XMAP215 is knocked down approximately 70%, this results in disruptions to all four functions, as described in the figure.
Ahmad,
Motor proteins regulate force interactions between microtubules and microfilaments in the axon.
2000, Pubmed
Ahmad,
Motor proteins regulate force interactions between microtubules and microfilaments in the axon.
2000,
Pubmed
Al-Bassam,
Regulation of microtubule dynamics by TOG-domain proteins XMAP215/Dis1 and CLASP.
2011,
Pubmed
,
Xenbase
Al-Bassam,
Fission yeast Alp14 is a dose-dependent plus end-tracking microtubule polymerase.
2012,
Pubmed
Applegate,
plusTipTracker: Quantitative image analysis software for the measurement of microtubule dynamics.
2011,
Pubmed
Ayaz,
A TOG:αβ-tubulin complex structure reveals conformation-based mechanisms for a microtubule polymerase.
2012,
Pubmed
Baas,
Force generation by cytoskeletal motor proteins as a regulator of axonal elongation and retraction.
2001,
Pubmed
Bamburg,
Assembly of microtubules at the tip of growing axons.
,
Pubmed
Becker,
Multiple isoforms of the high molecular weight microtubule associated protein XMAP215 are expressed during development in Xenopus.
2000,
Pubmed
,
Xenbase
Becker,
XMAP215, XKCM1, NuMA, and cytoplasmic dynein are required for the assembly and organization of the transient microtubule array during the maturation of Xenopus oocytes.
2003,
Pubmed
,
Xenbase
Brouhard,
XMAP215 is a processive microtubule polymerase.
2008,
Pubmed
,
Xenbase
Burnette,
Filopodial actin bundles are not necessary for microtubule advance into the peripheral domain of Aplysia neuronal growth cones.
2007,
Pubmed
Currie,
The microtubule lattice and plus-end association of Drosophila Mini spindles is spatially regulated to fine-tune microtubule dynamics.
2011,
Pubmed
Danuser,
Quantitative fluorescent speckle microscopy of cytoskeleton dynamics.
2006,
Pubmed
Gard,
A microtubule-associated protein from Xenopus eggs that specifically promotes assembly at the plus-end.
1987,
Pubmed
,
Xenbase
Hasaka,
Role of actin filaments in the axonal transport of microtubules.
2004,
Pubmed
Hur,
GSK3 controls axon growth via CLASP-mediated regulation of growth cone microtubules.
2011,
Pubmed
Keith,
Neurite elongation is blocked if microtubule polymerization is inhibited in PC12 cells.
1990,
Pubmed
Kumar,
GSK3beta phosphorylation modulates CLASP-microtubule association and lamella microtubule attachment.
2009,
Pubmed
Letourneau,
Differences in the organization of actin in the growth cones compared with the neurites of cultured neurons from chick embryos.
1983,
Pubmed
Lowery,
Neural Explant Cultures from Xenopus laevis.
2012,
Pubmed
,
Xenbase
Lowery,
The trip of the tip: understanding the growth cone machinery.
2009,
Pubmed
,
Xenbase
Lowery,
Parallel genetic and proteomic screens identify Msps as a CLASP-Abl pathway interactor in Drosophila.
2010,
Pubmed
Lu,
Initial neurite outgrowth in Drosophila neurons is driven by kinesin-powered microtubule sliding.
2013,
Pubmed
Mendoza,
Quantitative fluorescent speckle microscopy (QFSM) to measure actin dynamics.
2012,
Pubmed
Myers,
Antagonistic forces generated by cytoplasmic dynein and myosin-II during growth cone turning and axonal retraction.
2006,
Pubmed
Neukirchen,
Cytoplasmic linker proteins regulate neuronal polarization through microtubule and growth cone dynamics.
2011,
Pubmed
Santiago-Medina,
PAK-PIX interactions regulate adhesion dynamics and membrane protrusion to control neurite outgrowth.
2013,
Pubmed
,
Xenbase
Schaefer,
Filopodia and actin arcs guide the assembly and transport of two populations of microtubules with unique dynamic parameters in neuronal growth cones.
2002,
Pubmed
Shcherbo,
Far-red fluorescent tags for protein imaging in living tissues.
2009,
Pubmed
,
Xenbase
Shirasu-Hiza,
Identification of XMAP215 as a microtubule-destabilizing factor in Xenopus egg extract by biochemical purification.
2003,
Pubmed
,
Xenbase
Stepanova,
Visualization of microtubule growth in cultured neurons via the use of EB3-GFP (end-binding protein 3-green fluorescent protein).
2003,
Pubmed
Stone,
Global up-regulation of microtubule dynamics and polarity reversal during regeneration of an axon from a dendrite.
2010,
Pubmed
Suter,
Microtubule dynamics are necessary for SRC family kinase-dependent growth cone steering.
2004,
Pubmed
Tanaka,
Microtubule behavior in the growth cones of living neurons during axon elongation.
1991,
Pubmed
,
Xenbase
Tanaka,
The role of microtubule dynamics in growth cone motility and axonal growth.
1995,
Pubmed
,
Xenbase
Tournebize,
Control of microtubule dynamics by the antagonistic activities of XMAP215 and XKCM1 in Xenopus egg extracts.
2000,
Pubmed
,
Xenbase
Widlund,
XMAP215 polymerase activity is built by combining multiple tubulin-binding TOG domains and a basic lattice-binding region.
2011,
Pubmed
Wittmann,
Spatial regulation of CLASP affinity for microtubules by Rac1 and GSK3beta in migrating epithelial cells.
2005,
Pubmed
Zhou,
How actin filaments and microtubules steer growth cones to their targets.
2004,
Pubmed
van Breugel,
Stu2p, the budding yeast member of the conserved Dis1/XMAP215 family of microtubule-associated proteins is a plus end-binding microtubule destabilizer.
2003,
Pubmed
,
Xenbase
van der Vaart,
Microtubule plus-end tracking proteins SLAIN1/2 and ch-TOG promote axonal development.
2012,
Pubmed