Overexpression of the microtubule-binding protein CLIP-170 induces a +TIP network superstructure consistent with a biomolecular condensate - PubMed (original) (raw)
Overexpression of the microtubule-binding protein CLIP-170 induces a +TIP network superstructure consistent with a biomolecular condensate
Yueh-Fu O Wu et al. PLoS One. 2021.
Abstract
Proper regulation of microtubule (MT) dynamics is critical for cellular processes including cell division and intracellular transport. Plus-end tracking proteins (+TIPs) dynamically track growing MTs and play a key role in MT regulation. +TIPs participate in a complex web of intra- and inter- molecular interactions known as the +TIP network. Hypotheses addressing the purpose of +TIP:+TIP interactions include relieving +TIP autoinhibition and localizing MT regulators to growing MT ends. In addition, we have proposed that the web of +TIP:+TIP interactions has a physical purpose: creating a dynamic scaffold that constrains the structural fluctuations of the fragile MT tip and thus acts as a polymerization chaperone. Here we examine the possibility that this proposed scaffold is a biomolecular condensate (i.e., liquid droplet). Many animal +TIP network proteins are multivalent and have intrinsically disordered regions, features commonly found in biomolecular condensates. Moreover, previous studies have shown that overexpression of the +TIP CLIP-170 induces large "patch" structures containing CLIP-170 and other +TIPs; we hypothesized that these structures might be biomolecular condensates. To test this hypothesis, we used video microscopy, immunofluorescence staining, and Fluorescence Recovery After Photobleaching (FRAP). Our data show that the CLIP-170-induced patches have hallmarks indicative of a biomolecular condensate, one that contains +TIP proteins and excludes other known condensate markers. Moreover, bioinformatic studies demonstrate that the presence of intrinsically disordered regions is conserved in key +TIPs, implying that these regions are functionally significant. Together, these results indicate that the CLIP-170 induced patches in cells are phase-separated liquid condensates and raise the possibility that the endogenous +TIP network might form a liquid droplet at MT ends or other +TIP locations.
Conflict of interest statement
The authors have declared that no competing interests exist.
Figures
Fig 1. Conceptual models of the +TIP network.
(A) In the regulation-focused model of the +TIP network, interactions between +TIPs function primarily to activate autoinhibited +TIP proteins and/or enable +TIPs to localize to the plus end. (B) In a more physical model of the +TIP network, interactions between +TIP proteins form a cross-linked, dynamic scaffold that supports and stabilizes the fluctuating MT tips by promoting lateral binding of protofilaments [4]. This figure was created with BioRender [5].
Fig 2. CLIP-170 patches do not colocalize with membrane markers.
(A) Domain structures of the CLIP-170 constructs used in this study. (B) NIH3T3 or Cos-7 cells (as indicated) were transfected for 24 hr to overexpress GFP-CLIP-170, fixed with PFA, and observed by widefield fluorescence microscopy. Arrows point out examples of CLIP-170 patches as observed in cells at varied expression levels as indicated. The contrast of each image in a given row was adjusted to the same levels, except for high-level expression, where the main image was adjusted to allow the visualization of large and bright patch structures, and an inset provides a version normalized to match the rest of the row. (C) As an example of the failure of membrane markers to colocalize with patches, NIH3T3 cells were transfected with GFP-CLIP-170 for 24 hours. Rhodamine-DHPE or Rhodamine-PE was used as a generic membrane label to stain lipid membranes. Live cells were observed by widefield fluorescence microscopy. See S2 Fig in S5 File for colocalizations with additional membrane markers. Scale bar: 10 μm.
Fig 3. Dynamic behaviors of CLIP-170 patches in vivo.
NIH3T3 cells were transfected to overexpress GFP-CLIP-170, and the behavior of GFP-CLIP-170 in vivo was recorded by confocal microscopy after 24–27 hr of transfection. Red box: An example of an apparent elastic deformation of a patch, following by fission (blow-up of this region is shown below). Arrows indicate examples of patch fusion (magenta), a photobleached site (green, relevant to discussions below), and a typical comet-shaped +TIP localization on a MT tip (yellow).
Fig 4. CLIP-170 patches have selective properties.
NIH3T3 cells were transfected with full-length CLIP-170 for 24 hr before fixation and staining as indicated. For the EB1, p150, and YB1 labeling, cells were fixed with methanol; for LIS1 and mDia2, cells were fixed with PFA. The contrast of all images in a given row was set to the same levels. These data show that EB1, p150, LIS-1, and mDia2 were included in the CLIP-170 patches; YB1 is an example of a molecule that was excluded from the patches. See S3 Fig in S5 File for images of colocalizations with other molecules, and see Table 1 for a summarized list. Scale bar: 10 μm.
Fig 5. MTs are not required for CLIP-170 patch formation and maintenance.
(A) Experimental design diagram. (B) NIH3T3 cells were transfected with full-length CLIP-170, and cells were treated with 5 μM nocodazole (Noc) after 8 hr (Set 1) or 23 hr (Set 2) transfection at 37°C. Cells were fixed with methanol after 24 hr transfection, probed with antibodies against molecules of interest (as indicated), and observed by widefield fluorescence microscopy. After the nocodazole treatment, tubulin and EB1 colocalized with CLIP-170 patches, while YB1 did not colocalize with the patches. Each image was normalized separately to allow the visualization of the different staining and patches. Scale bar: 10 μm.
Fig 6. FRAP analysis of the dynamics of CLIP-170 patches.
(A) Example of a partially photobleached CLIP-170 patch in an NIH3T3 cell. (B) The time course plot for the FRAP spot in (A). This panel is one example representing the fluorescence recovery of one bleaching site in one CLIP-170 patch to show how we processed the data. The intensity values were normalized against the average mean intensity before bleaching. (C, D, E) Quantification of the t1/2, mobile fraction, and immobile fraction. A total of 30 condensates from 30 separate cells were photobleached, and their recovery profiles were fitted to the exponential recovery equation (see Methods for details). “All” indicates the summation of data without regard to droplet size. In addition, we analyzed the droplets as separated into small, medium, and large groups (see S6 Fig in S5 File for more information about how this separation was performed). (C) provides a numerical summary of the t1/2, mobile fraction, and immobile fraction. Panel (D) provides a box plot of the t1/2 of each size group, while panel (E) provides a bar graph of the fractions of mobile and immobile protein. n = 14 (Small), 11 (Medium), and 5 (Large), for a total of 30 droplets. The error bars show the standard deviation; other aspects of the box plot are as generated by the default Excel Box and Whisker plot function. N.S. = Not Significant (there were no significant differences between any of these groups).
Fig 7. Analysis of coiled-coil domains and IDRs for human CLIP-170 (AAA35693.1).
(Top) CLIP-170 domain structure summary. The N-terminal domain includes the 2 CAP-Gly domains and 3 serine-rich regions. The coiled-coil domain is labeled in orange, and its boundaries are indicated with two red lines. The C-terminal domain contains two zinc-knuckle motifs (yellow). (Middle) Analysis of coiled-coil propensity as predicted by COILS. The colored lines represent the probability that a region assumes a coiled-coil conformation, as assessed for different windows (7AA, blue; 14 AA, orange; 21 AA yellow), with 1 on the Y-axis indicating 100% likelihood. (Bottom) IDR predictions (Y-axis) as function of amino acid position (X-axis), with 1 indicating 100% likelihood. The purple line represents the probability of IDRs as predicted by Espritz, with red arrows indicating the 10 regions predicted to be disordered. The grey areas indicate the IDRs as predicted by MobiDB-lite. The green line represents the probability of IDRs as predicted by IUPred2A, and the light green indicates the probability of 0.5.
Fig 8. Analysis of coiled-coil and IDRs in CLIP-170 relatives from a range of different species.
For each organism listed, one representative CLIP-170 sequence was selected for analysis, the results of which are shown in three images. The set of upper images provides the domain structure for each sequence, with colors as indicated: blue: CAP-Gly domains; orange: Coiled-coil regions; pink: FEED domain; yellow: Zinc-knuckle motifs. The sets of middle and lower images respectively provide the probabilities that a region is coiled-coil (as predicted by COILS) or IDR (as predicted by Espritz). For both analyses, 1 indicates 100% likelihood and * indicates the end of the sequence.
Fig 9. Analyses of IDRs in members of the human +TIP network.
The Y-axis provides the probability of IDRs as a function of amino acid position (X-axis); 1 indicates 100% likelihood. The purple line provides the data for Espritz; shaded areas are the disordered regions predicted by MobiDB-lite. See Materials and Methods for the accession numbers.
Fig 10. Proposed polymerization chaperone model.
(A) In absence of the +TIP network, tubulin subunits arrive and leave quickly because most newly attached subunits are initially in the laterally unbonded regions of the tip protofilaments. The result is that MTs grow relatively slowly because most subunits detach before being incorporated into the laterally bound region of the MT. (B) In the presence of the proposed +TIP network liquid droplet, the web of interactions between the +TIPs creates a dynamic stocking-like structure that tracks the tip by diffusing on the potential energy gradient created by the preferential affinity of some tip components (most notably EB1) for tip-specific tubulin conformations. As the droplet moves, it exerts a force on the tip that promotes the zipping up of lateral bonds between protofilaments. This effect in turn increases the likelihood that newly arrived subunits are incorporated into the lattice and enables the MT to grow faster, as seen in vivo or with mixtures of +TIPs in vitro [4]. This figure was created with BioRender [5].
References
- BioRender. 2020. Available from: https://biorender.com/.
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This research was funded by a fellowship from the American Heart Association https://www.heart.org/en (#17PRE33670896) to YOW and a grant from the National Science Foundation https://www.nsf.gov/ (MCB #1817966) to HVG. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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