Phase separation on microtubules: from droplet formation to cellular function?

Liquid–liquid phase separation (LLPS) is a common phenomenon observed for microtubule-binding proteins expressed recombinantly in vitro, or overexpressed in cells.LLPS of microtubule-binding proteins in vitro can be driven by very different types of interactions, involving intrinsically disordered regions and folded domains.Binding to microtubules can promote formation of protein condensates.Condensates of tubulin-binding proteins can potentially promote microtubule nucleation and accelerate microtubule elongation.Condensate formation by the same or homologous proteins strongly depends on the species, cell type, or cell cycle phase.Conclusive evidence that LLPS occurs at physiological conditions in cells is often missing. LLPS (see Glossary) is a process of demixing of two immiscible or semi-miscible liquids, often illustrated through the 'vinegar in oil' analogy. LLPS has been proposed to be the mechanism behind the formation of membraneless cell compartments, often termed 'biomolecular condensates', including nucleoli, P-bodies, and stress granules, where specific macromolecules such as proteins or nucleic acids are concentrated (reviewed in [1.Banani S.F. et al.Biomolecular condensates: organizers of cellular biochemistry.Nat. Rev. Mol. Cell Biol. 2017; 18: 285-298Crossref PubMed Scopus (2457) Google Scholar,2.Hyman A.A. et al.Liquid-liquid phase separation in biology.Annu. Rev. Cell Dev. Biol. 2014; 30: 39-58Crossref PubMed Google Scholar]). In this review, we will focus on microtubule-binding proteins and protein complexes and discuss the evidence that they form non-stoichiometric condensates through LLPS. We will use the term LLPS broadly, as is currently common in cell biology literature, although it is clear that the term is often used to describe phases that are not simple liquids [3.Mittag T. Pappu R.V. A conceptual framework for understanding phase separation and addressing open questions and challenges.Mol. Cell. 2022; 82: 2201-2214Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar]. What interactions drive proteins into the condensed phase? Homo- and heterotypic protein–protein interactions can depend on the binding between folded domains, between a folded domain and a linear motif, or the interactions between intrinsically disordered protein regions (IDRs) [4.Musacchio A. On the role of phase separation in the biogenesis of membraneless compartments.EMBO J. 2022; 41e109952Crossref PubMed Scopus (38) Google Scholar,5.Dignon G.L. et al.Biomolecular phase separation: from molecular driving forces to macroscopic properties.Annu. Rev. Phys. Chem. 2020; 71: 53-75Crossref PubMed Scopus (182) Google Scholar] (Figure 1A ). Many proteins that exhibit LLPS in vitro carry IDRs; presence of IDRs correlates with the ability of these proteins to phase-separate [6.Vernon R.M. Forman-Kay J.D. First-generation predictors of biological protein phase separation.Curr. Opin. Struct. Biol. 2019; 58: 88-96Crossref PubMed Scopus (71) Google Scholar]. IDRs often display poor sequence conservation, leading to the idea that formation of mesoscale compartments by LLPS is driven by low-affinity interactions between IDRs and relies on physical properties of amino acids rather than sequence-encoded specific, high-affinity interactions [7.Hyman A.A. Brangwynne C.P. Beyond stereospecificity: liquids and mesoscale organization of cytoplasm.Dev. Cell. 2011; 21: 14-16Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar,8.Wang J. et al.A molecular grammar governing the driving forces for phase separation of prion-like RNA binding proteins.Cell. 2018; 174: 688-699Abstract Full Text Full Text PDF PubMed Scopus (855) Google Scholar]. However, in principle, sequence-specific, high affinity interactions involving folded domains might also promote formation of protein condensates [4.Musacchio A. On the role of phase separation in the biogenesis of membraneless compartments.EMBO J. 2022; 41e109952Crossref PubMed Scopus (38) Google Scholar]. An important consequence of LLPS is 'concentration buffering': changing the amount of a phase-separating protein leads to the change in the volume of the condensed phase, while the protein's concentration in the 'dilute phase' remains constant (Figure 1A). A simple way to induce protein condensation is therefore to increase its concentration. In vivo, this can be achieved by overexpressing the protein of interest and observing formation of droplets that can fuse over time. When working with purified proteins in vitro, inert 'crowding agents' such as polyethylene glycol (PEG) can be added to deplete the solvent volume accessible to proteins and increase their local concentration to promote LLPS. However, the relevance of specific crowding agents in mimicking cellular environment is limited, because cellular components can both promote and inhibit condensate formation and overall can have major effects on phase separation of a particular protein [9.Riback J.A. et al.Composition-dependent thermodynamics of intracellular phase separation.Nature. 2020; 581: 209-214Crossref PubMed Scopus (253) Google Scholar]. These approaches have been routinely used to observe LLPS of numerous proteins, but in fact, studying LLPS of a given protein is only relevant if it happens at the physiological concentration. Given how easily overexpression can result in condensate formation, tight control of expression levels is therefore essential, but, unfortunately, often omitted in studies reporting LLPS (reviewed in [10.McSwiggen D.T. et al.Evaluating phase separation in live cells: diagnosis, caveats, and functional consequences.Genes Dev. 2019; 33: 1619-1634Crossref PubMed Scopus (290) Google Scholar]). Furthermore, as the name implies, for the LLPS mechanism to hold true, the condensed phase should remain liquid and exchanging with the dilute phase. Evidence to this is provided either qualitatively, by demonstrating 'fusion' of protein condensates, or quantitatively, by measuring fluorescence recovery after photobleaching (FRAP). However, for microscopic condensates, fusion can be easily confused with co-localization of sub-diffraction protein foci and fluorescence recovery can be influenced by interactions distinct from LLPS, such as transient binding to a scaffold. In many cases, protein droplets are not exchanging with the solution and are described as 'hardening', or 'gelating'. In these conditions, it may be difficult to determine whether their formation occurs through LLPS or aggregation; alternatively, the concentration of molecules in the dilute phase may be simply very low to observe exchange with the condensed phase. Reagents widely used to distinguish LLPS from aggregation are derivatives of hexanediol, which disrupt certain types of hydrophobic interactions [11.Ribbeck K. Görlich D. The permeability barrier of nuclear pore complexes appears to operate via hydrophobic exclusion.EMBO J. 2002; 21: 2664-2671Crossref PubMed Scopus (431) Google Scholar] and can affect LLPS of various proteins both in vitro and in vivo, while having little effect on some non-LLPS interactions; however, these reagents are cytotoxic [12.Kroschwald S. et al.Hexanediol: a chemical probe to investigate the material properties of membrane-less compartments.Matters. 2017; (Published online May 22, 2017. https://doi.org/10.19185/matters.201702000010)Crossref Google Scholar] and thus performing appropriate controls is very difficult [13.Düster R. et al.1,6-Hexanediol, commonly used to dissolve liquid–liquid phase separated condensates, directly impairs kinase and phosphatase activities.J. Biol. Chem. 2021; 296100260Abstract Full Text Full Text PDF PubMed Google Scholar] (Box 1).Box 1Challenges in linking results of in vitro reconstitution to cellular functionSeveral common strategies are used to link formation of protein condensates in vitro to the existence of LLPS-mediated structures in vivo and to the conclusion that LLPS is functionally important:(i)Deletion/mutation approach. Formation (or lack thereof) of spherical droplets or condensates of full-length and mutant proteins in vitro is correlated to localization of the same protein constructs in cells. This approach is limited by the fact that deleted/mutated protein regions might mediate interactions that are independent of LLPS. This approach can be potentially improved by generating protein constructs that lack LLPS properties, which may show some redundancy, but retain other relevant interactions (e.g., folding properties and binding to partners), and vice versa [4.Musacchio A. On the role of phase separation in the biogenesis of membraneless compartments.EMBO J. 2022; 41e109952Crossref PubMed Scopus (38) Google Scholar].(ii)Protein overexpression with or without additional oligomerization domains such as Cry2 [100.Shin Y. et al.Spatiotemporal control of intracellular phase transitions using light-activated optoDroplets.Cell. 2017; 168: 159-171Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar]. This approach is technically less challenging than studying endogenously tagged proteins and can be very useful for studying biophysical aspects of phase separation in cells, but it strongly promotes condensate formation and is by itself not informative about the behavior of endogenous, non-tagged proteins (reviewed in [10.McSwiggen D.T. et al.Evaluating phase separation in live cells: diagnosis, caveats, and functional consequences.Genes Dev. 2019; 33: 1619-1634Crossref PubMed Scopus (290) Google Scholar]).(iii)Observation of 'liquid-like behavior' and 'fusion events' between protein condensates in cells. This approach can provide direct evidence for the liquid-like nature of protein oligomers, but distinguishing fusion from co-localization can be challenging for diffraction-limited foci, even using super-resolution microscopy [72.Meier S.M. et al.Multivalency ensures persistence of a +TIP body at specialized microtubule ends.Nat. Cell Biol. 2023; 25: 56-67Crossref PubMed Scopus (4) Google Scholar, 73.Miesch J. et al.Phase separation of +TIP-networks regulates microtubule dynamics.bioRxiv. 2022; (March 3, 2022. https://doi.org/10.1101/2021.09.13.459419)Google Scholar, 74.Song X. et al.Phase separation of EB1 guides microtubule plus-end dynamics.Nat. Cell Biol. 2023; 25: 79-91Crossref PubMed Scopus (3) Google Scholar].(iv)FRAP to test the exchange between condensed and dilute phases. Low recovery rates could be interpreted as formation of stable condensates [18.Woodruff J.B. et al.The centrosome is a selective condensate that nucleates microtubules by concentrating tubulin.Cell. 2017; 169: 1066-1077Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar,20.Sahu S. et al.Spatially controlled microtubule nucleation and organization from crosslinker MAP65 condensates.bioRxiv. 2022; (Published online October 23, 2002. https://doi.org/10.1101/2022.10.23.513406)Google Scholar,21.Trivedi P. et al.The inner centromere is a biomolecular condensate scaffolded by the chromosomal passenger complex.Nat. Cell Biol. 2019; 21: 1127-1137Crossref PubMed Scopus (48) Google Scholar,48.Tan R. et al.Microtubules gate tau condensation to spatially regulate microtubule functions.Nat. Cell Biol. 2019; 21: 1078-1085Crossref PubMed Google Scholar,72.Meier S.M. et al.Multivalency ensures persistence of a +TIP body at specialized microtubule ends.Nat. Cell Biol. 2023; 25: 56-67Crossref PubMed Scopus (4) Google Scholar, 73.Miesch J. et al.Phase separation of +TIP-networks regulates microtubule dynamics.bioRxiv. 2022; (March 3, 2022. https://doi.org/10.1101/2021.09.13.459419)Google Scholar, 74.Song X. et al.Phase separation of EB1 guides microtubule plus-end dynamics.Nat. Cell Biol. 2023; 25: 79-91Crossref PubMed Scopus (3) Google Scholar], while high recovery rates, as formation of liquid droplets [18.Woodruff J.B. et al.The centrosome is a selective condensate that nucleates microtubules by concentrating tubulin.Cell. 2017; 169: 1066-1077Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar,20.Sahu S. et al.Spatially controlled microtubule nucleation and organization from crosslinker MAP65 condensates.bioRxiv. 2022; (Published online October 23, 2002. https://doi.org/10.1101/2022.10.23.513406)Google Scholar,22.von Appen A. et al.LEM2 phase separation promotes ESCRT-mediated nuclear envelope reformation.Nature. 2020; 582: 115-118Crossref PubMed Scopus (65) Google Scholar,38.King M.R. Petry S. Phase separation of TPX2 enhances and spatially coordinates microtubule nucleation.Nat. Commun. 2020; 11: 270Crossref PubMed Scopus (80) Google Scholar,72.Meier S.M. et al.Multivalency ensures persistence of a +TIP body at specialized microtubule ends.Nat. Cell Biol. 2023; 25: 56-67Crossref PubMed Scopus (4) Google Scholar, 73.Miesch J. et al.Phase separation of +TIP-networks regulates microtubule dynamics.bioRxiv. 2022; (March 3, 2022. https://doi.org/10.1101/2021.09.13.459419)Google Scholar, 74.Song X. et al.Phase separation of EB1 guides microtubule plus-end dynamics.Nat. Cell Biol. 2023; 25: 79-91Crossref PubMed Scopus (3) Google Scholar,85.Zhang M. et al.Molecular organization of the early stages of nucleosome phase separation visualized by cryo-electron tomography.Mol. Cell. 2022; 82: 3000-3014Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar,94.Zhang M. et al.SKAP interacts with Aurora B to guide end-on capture of spindle microtubules via phase separation.J. Mol. Cell Biol. 2021; 13: 841-852Crossref Scopus (1) Google Scholar], often within the same study. However, LLPS-independent interactions, such as binding between proteins, or interactions with a scaffold can also influence mobility of proteins [10.McSwiggen D.T. et al.Evaluating phase separation in live cells: diagnosis, caveats, and functional consequences.Genes Dev. 2019; 33: 1619-1634Crossref PubMed Scopus (290) Google Scholar]. Altogether, FRAP provides no information on the material properties of a particular cellular structure and may not be informative when the protein concentration in the dilute phase is very low.(v)Use of hexanediol to dissolve condensates. Hexanediol has been proposed to selectively dissolve membraneless organelles formed by LLPS while leaving protein aggregates intact [12.Kroschwald S. et al.Hexanediol: a chemical probe to investigate the material properties of membrane-less compartments.Matters. 2017; (Published online May 22, 2017. https://doi.org/10.19185/matters.201702000010)Crossref Google Scholar]. However, recent evidence points to side effects of this reagent on various cellular processes [13.Düster R. et al.1,6-Hexanediol, commonly used to dissolve liquid–liquid phase separated condensates, directly impairs kinase and phosphatase activities.J. Biol. Chem. 2021; 296100260Abstract Full Text Full Text PDF PubMed Google Scholar,101.Ulianov S.V. et al.Suppression of liquid–liquid phase separation by 1,6-hexanediol partially compromises the 3D genome organization in living cells.Nucleic Acids Res. 2021; 49: 10524-10541Crossref PubMed Scopus (0) Google Scholar]. Furthermore, hexanediol preferentially disrupts hydrophobic interactions, whereas LLPS can also be driven by, for example, electrostatic interactions that are hexanediol-insensitive.(vi)Analysis by electron microscopy and tomography. This technique can provide insight into the internal organization and dimensions of protein droplets and oligomers at the scale inaccessible to light microscopy [18.Woodruff J.B. et al.The centrosome is a selective condensate that nucleates microtubules by concentrating tubulin.Cell. 2017; 169: 1066-1077Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar,39.Setru S.U. et al.A hydrodynamic instability drives protein droplet formation on microtubules to nucleate branches.Nat. Phys. 2021; 17: 493-498Crossref PubMed Scopus (6) Google Scholar,65.Maan R. et al.Multivalent interactions facilitate motor-dependent protein accumulation at growing microtubule plus-ends.Nat. Cell Biol. 2023; 25: 68-78Crossref PubMed Scopus (3) Google Scholar,86.Jakobi A.J. et al.Structural basis of p62/SQSTM1 helical filaments and their role in cellular cargo uptake.Nat. Commun. 2020; 11: 440Crossref PubMed Scopus (44) Google Scholar]. However, interpreting flexible protein structures in crowded cellular environment is challenging and, even in the case of in vitro reconstituted oligomerization, these observations remain qualitative and do not allow to clearly distinguish LLPS from other protein–protein interactions [65.Maan R. et al.Multivalent interactions facilitate motor-dependent protein accumulation at growing microtubule plus-ends.Nat. Cell Biol. 2023; 25: 68-78Crossref PubMed Scopus (3) Google Scholar]. Several common strategies are used to link formation of protein condensates in vitro to the existence of LLPS-mediated structures in vivo and to the conclusion that LLPS is functionally important:(i)Deletion/mutation approach. Formation (or lack thereof) of spherical droplets or condensates of full-length and mutant proteins in vitro is correlated to localization of the same protein constructs in cells. This approach is limited by the fact that deleted/mutated protein regions might mediate interactions that are independent of LLPS. This approach can be potentially improved by generating protein constructs that lack LLPS properties, which may show some redundancy, but retain other relevant interactions (e.g., folding properties and binding to partners), and vice versa [4.Musacchio A. On the role of phase separation in the biogenesis of membraneless compartments.EMBO J. 2022; 41e109952Crossref PubMed Scopus (38) Google Scholar].(ii)Protein overexpression with or without additional oligomerization domains such as Cry2 [100.Shin Y. et al.Spatiotemporal control of intracellular phase transitions using light-activated optoDroplets.Cell. 2017; 168: 159-171Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar]. This approach is technically less challenging than studying endogenously tagged proteins and can be very useful for studying biophysical aspects of phase separation in cells, but it strongly promotes condensate formation and is by itself not informative about the behavior of endogenous, non-tagged proteins (reviewed in [10.McSwiggen D.T. et al.Evaluating phase separation in live cells: diagnosis, caveats, and functional consequences.Genes Dev. 2019; 33: 1619-1634Crossref PubMed Scopus (290) Google Scholar]).(iii)Observation of 'liquid-like behavior' and 'fusion events' between protein condensates in cells. This approach can provide direct evidence for the liquid-like nature of protein oligomers, but distinguishing fusion from co-localization can be challenging for diffraction-limited foci, even using super-resolution microscopy [72.Meier S.M. et al.Multivalency ensures persistence of a +TIP body at specialized microtubule ends.Nat. Cell Biol. 2023; 25: 56-67Crossref PubMed Scopus (4) Google Scholar, 73.Miesch J. et al.Phase separation of +TIP-networks regulates microtubule dynamics.bioRxiv. 2022; (March 3, 2022. https://doi.org/10.1101/2021.09.13.459419)Google Scholar, 74.Song X. et al.Phase separation of EB1 guides microtubule plus-end dynamics.Nat. Cell Biol. 2023; 25: 79-91Crossref PubMed Scopus (3) Google Scholar].(iv)FRAP to test the exchange between condensed and dilute phases. Low recovery rates could be interpreted as formation of stable condensates [18.Woodruff J.B. et al.The centrosome is a selective condensate that nucleates microtubules by concentrating tubulin.Cell. 2017; 169: 1066-1077Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar,20.Sahu S. et al.Spatially controlled microtubule nucleation and organization from crosslinker MAP65 condensates.bioRxiv. 2022; (Published online October 23, 2002. https://doi.org/10.1101/2022.10.23.513406)Google Scholar,21.Trivedi P. et al.The inner centromere is a biomolecular condensate scaffolded by the chromosomal passenger complex.Nat. Cell Biol. 2019; 21: 1127-1137Crossref PubMed Scopus (48) Google Scholar,48.Tan R. et al.Microtubules gate tau condensation to spatially regulate microtubule functions.Nat. Cell Biol. 2019; 21: 1078-1085Crossref PubMed Google Scholar,72.Meier S.M. et al.Multivalency ensures persistence of a +TIP body at specialized microtubule ends.Nat. Cell Biol. 2023; 25: 56-67Crossref PubMed Scopus (4) Google Scholar, 73.Miesch J. et al.Phase separation of +TIP-networks regulates microtubule dynamics.bioRxiv. 2022; (March 3, 2022. https://doi.org/10.1101/2021.09.13.459419)Google Scholar, 74.Song X. et al.Phase separation of EB1 guides microtubule plus-end dynamics.Nat. Cell Biol. 2023; 25: 79-91Crossref PubMed Scopus (3) Google Scholar], while high recovery rates, as formation of liquid droplets [18.Woodruff J.B. et al.The centrosome is a selective condensate that nucleates microtubules by concentrating tubulin.Cell. 2017; 169: 1066-1077Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar,20.Sahu S. et al.Spatially controlled microtubule nucleation and organization from crosslinker MAP65 condensates.bioRxiv. 2022; (Published online October 23, 2002. https://doi.org/10.1101/2022.10.23.513406)Google Scholar,22.von Appen A. et al.LEM2 phase separation promotes ESCRT-mediated nuclear envelope reformation.Nature. 2020; 582: 115-118Crossref PubMed Scopus (65) Google Scholar,38.King M.R. Petry S. Phase separation of TPX2 enhances and spatially coordinates microtubule nucleation.Nat. Commun. 2020; 11: 270Crossref PubMed Scopus (80) Google Scholar,72.Meier S.M. et al.Multivalency ensures persistence of a +TIP body at specialized microtubule ends.Nat. Cell Biol. 2023; 25: 56-67Crossref PubMed Scopus (4) Google Scholar, 73.Miesch J. et al.Phase separation of +TIP-networks regulates microtubule dynamics.bioRxiv. 2022; (March 3, 2022. https://doi.org/10.1101/2021.09.13.459419)Google Scholar, 74.Song X. et al.Phase separation of EB1 guides microtubule plus-end dynamics.Nat. Cell Biol. 2023; 25: 79-91Crossref PubMed Scopus (3) Google Scholar,85.Zhang M. et al.Molecular organization of the early stages of nucleosome phase separation visualized by cryo-electron tomography.Mol. Cell. 2022; 82: 3000-3014Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar,94.Zhang M. et al.SKAP interacts with Aurora B to guide end-on capture of spindle microtubules via phase separation.J. Mol. Cell Biol. 2021; 13: 841-852Crossref Scopus (1) Google Scholar], often within the same study. However, LLPS-independent interactions, such as binding between proteins, or interactions with a scaffold can also influence mobility of proteins [10.McSwiggen D.T. et al.Evaluating phase separation in live cells: diagnosis, caveats, and functional consequences.Genes Dev. 2019; 33: 1619-1634Crossref PubMed Scopus (290) Google Scholar]. Altogether, FRAP provides no information on the material properties of a particular cellular structure and may not be informative when the protein concentration in the dilute phase is very low.(v)Use of hexanediol to dissolve condensates. Hexanediol has been proposed to selectively dissolve membraneless organelles formed by LLPS while leaving protein aggregates intact [12.Kroschwald S. et al.Hexanediol: a chemical probe to investigate the material properties of membrane-less compartments.Matters. 2017; (Published online May 22, 2017. https://doi.org/10.19185/matters.201702000010)Crossref Google Scholar]. However, recent evidence points to side effects of this reagent on various cellular processes [13.Düster R. et al.1,6-Hexanediol, commonly used to dissolve liquid–liquid phase separated condensates, directly impairs kinase and phosphatase activities.J. Biol. Chem. 2021; 296100260Abstract Full Text Full Text PDF PubMed Google Scholar,101.Ulianov S.V. et al.Suppression of liquid–liquid phase separation by 1,6-hexanediol partially compromises the 3D genome organization in living cells.Nucleic Acids Res. 2021; 49: 10524-10541Crossref PubMed Scopus (0) Google Scholar]. Furthermore, hexanediol preferentially disrupts hydrophobic interactions, whereas LLPS can also be driven by, for example, electrostatic interactions that are hexanediol-insensitive.(vi)Analysis by electron microscopy and tomography. This technique can provide insight into the internal organization and dimensions of protein droplets and oligomers at the scale inaccessible to light microscopy [18.Woodruff J.B. et al.The centrosome is a selective condensate that nucleates microtubules by concentrating tubulin.Cell. 2017; 169: 1066-1077Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar,39.Setru S.U. et al.A hydrodynamic instability drives protein droplet formation on microtubules to nucleate branches.Nat. Phys. 2021; 17: 493-498Crossref PubMed Scopus (6) Google Scholar,65.Maan R. et al.Multivalent interactions facilitate motor-dependent protein accumulation at growing microtubule plus-ends.Nat. Cell Biol. 2023; 25: 68-78Crossref PubMed Scopus (3) Google Scholar,86.Jakobi A.J. et al.Structural basis of p62/SQSTM1 helical filaments and their role in cellular cargo uptake.Nat. Commun. 2020; 11: 440Crossref PubMed Scopus (44) Google Scholar]. However, interpreting flexible protein structures in crowded cellular environment is challenging and, even in the case of in vitro reconstituted oligomerization, these observations remain qualitative and do not allow to clearly distinguish LLPS from other protein–protein interactions [65.Maan R. et al.Multivalent interactions facilitate motor-dependent protein accumulation at growing microtubule plus-ends.Nat. Cell Biol. 2023; 25: 68-78Crossref PubMed Scopus (3) Google Scholar]. Microtubules are cytoskeletal filaments with lattice-like walls built from globular tubulin subunits, which have negatively charged disordered C-terminal tails extending into solution (Figure 1B). Highly ordered polymeric structures of microtubule lattices can concentrate microtubule-binding proteins through specific interactions that depend on folded domains and/or IDRs. Additionally, some microtubule-binding proteins have increased affinity for microtubule ends, leading to their accumulation in even smaller volumes. This increased affinity can result from preferential binding to bent tubulin protofilaments, to specific tubulin conformations associated with certain states of GTP hydrolysis, or to tubulin surfaces or interfaces that are only exposed at microtubule ends [14.Akhmanova A. Steinmetz M.O. Control of microtubule organization and dynamics: two ends in the limelight.Nat. Rev. Mol. Cell Biol. 2015; 16: 711-726Crossref PubMed Scopus (561) Google Scholar,15.Roostalu J. et al.The speed of GTP hydrolysis determines GTP cap size and controls microtubule stability.eLife. 2020; 9e51992Crossref PubMed Scopus (40) Google Scholar]. Furthermore, many microtubule-binding proteins can associate with each other, forming complex multivalent interaction networks [14.Akhmanova A. Steinmetz M.O. Control of microtubule organization and dynamics: two ends in the limelight.Nat. Rev. Mol. Cell Biol. 2015; 16: 711-726Crossref PubMed Scopus (561) Google Scholar]. Together, all these interactions can trigger formation of microtubule surface-bound protein condensates or liquid droplets, held together by low-affinity interactions that cannot be observed in cytosol but require local enrichment provided by microtubule lattices and ends (Figure 1C; discussed in [16.Mitchison T.J. Beyond Langmuir: surface-bound macromolecule condensates.Mol. Biol. Cell. 2020; 31: 2502-2508Crossref PubMed Google Scholar]). Condensates of microtubule-binding proteins can potentially help to locally control microtubule stability and dynamics, promote interactions with other cellular structures, or generate functionally different microtubule subsets that can be recognized by motors responsible for intracellular transport. Furthermore, the reverse process of concentrating tubulin by a droplet of a microtubule-binding protein can trigger microtubule nucleation or accelerate microtubule polymerization. Recent publications propose involvement of LLPS in the formation and function of various microtubule-based cellular structures (Figure 1D). Next, we consider different examples of microtubule-binding proteins with demonstrated ability to undergo LLPS in vitro and/or in cells and discuss the potential functional relevance of these observations. The rate-limiting step in the initiation of microtubule polymerization is the formation of the primary nucleus or template (reviewed in [17.Roostalu J. Surrey T. Microtubule nucleation: beyond the template.Nat. Rev. Mol. Cell Biol. 2017; 18: 702-710Crossref PubMed Scopus (109) Google Scholar]). Therefore, local concentration of tubulin in a small volume due to a high density of tubulin-binding molecules should facilitate nucleation of microtubules. With this mechanism in mind, the centrosome, a major microtubule-organizing center (MTOC) in animal cells, has been proposed to be a phase-separated condensate that concentrates tubulin-binding molecules and tubulin [18.Woodruff J.B. et al.The centrosome is a selective condensate that nucleates microtubules by concentrating tubulin.Cell. 2017; 169: 1066-1077Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar]. This idea is supported by the ability of a key player in centrosome formation in Caenorhabditis elegans, a coiled coil scaffold SPD-5, to undergo LLPS and subsequently harden into gel-like condensates that can concentrate worm homologs of microtubule and tubulin-binding proteins TPX2 and XMAP215, accumulate tubulin, and nucleate microtubules in vitro [18.Woodruff J.B. et al.The centrosome is a selective condensate that nucleates microtubules by concentrating tubulin.Cell. 2017; 169: 1066-1077Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar]. It should be noted, however, that microtubule-nucleating properties were