Organization of microtubule plus-end dynamics by phase separation in mitosis.

In eukaryotes, microtubule polymers are essential for cellular plasticity and fate decisions. End-binding (EB) proteins serve as scaffolds for orchestrating microtubule polymer dynamics and are essential for cellular dynamics and chromosome segregation in mitosis. Here, we show that EB1 forms molecular condensates with TIP150 and MCAK through liquid-liquid phase separation to compartmentalize the kinetochore-microtubule plus-end machinery, ensuring accurate kinetochore-microtubule interactions during chromosome segregation in mitosis. Perturbation of EB1-TIP150 polymer formation by a competing peptide prevents phase separation of the EB1-mediated complex and chromosome alignment at the metaphase equator in both cultured cells and Drosophila embryos. Lys220 of EB1 is dynamically acetylated by p300/CBP-associated factor in early mitosis, and persistent acetylation at Lys220 attenuates phase separation of the EB1-mediated complex, dissolves droplets in vitro, and harnesses accurate chromosome segregation. Our data suggest a novel framework for understanding the organization and regulation of eukaryotic spindle for accurate chromosome segregation in mitosis.

Long way up: rethink diseases in light of phase separation and phase transition.

Biomolecular condensation, driven by multivalency, serves as a fundamental mechanism within cells, facilitating the formation of distinct compartments, including membraneless organelles that play essential roles in various cellular processes. Perturbations in the delicate equilibrium of condensation, whether resulting in gain or loss of phase separation, have robustly been associated with cellular dysfunction and physiological disorders. As ongoing research endeavors wholeheartedly embrace this newly acknowledged principle, a transformative shift is occurring in our comprehension of disease. Consequently, significant strides have been made in unraveling the profound relevance and potential causal connections between abnormal phase separation and various diseases. This comprehensive review presents compelling recent evidence that highlight the intricate associations between aberrant phase separation and neurodegenerative diseases, cancers, and infectious diseases. Additionally, we provide a succinct summary of current efforts and propose innovative solutions for the development of potential therapeutics to combat the pathological consequences attributed to aberrant phase separation.

Phase separation of EB1 guides microtubule plus-end dynamics.

In eukaryotes, end-binding (EB) proteins serve as a hub for orchestrating microtubule dynamics and are essential for cellular dynamics and organelle movements. EB proteins modulate structural transitions at growing microtubule ends by recognizing and promoting an intermediate state generated during GTP hydrolysis. However, the molecular mechanisms and physiochemical properties of the EB1 interaction network remain elusive. Here we show that EB1 formed molecular condensates through liquid-liquid phase separation (LLPS) to constitute the microtubule plus-end machinery. EB1 LLPS is driven by multivalent interactions among different segments, which are modulated by charged residues in the linker region. Phase-separated EB1 provided a compartment for enriching tubulin dimers and other plus-end tracking proteins. Real-time imaging of chromosome segregation in HeLa cells expressing LLPS-deficient EB1 mutants revealed the importance of EB1 LLPS dynamics in mitotic chromosome movements. These findings demonstrate that EB1 forms a distinct physical and biochemical membraneless-organelle via multivalent interactions that guide microtubule dynamics.

Myosin 1D and the branched actin network control the condensation of p62 bodies.

Biomolecular condensation driven by liquid-liquid phase separation (LLPS) is key to assembly of membraneless organelles in numerous crucial pathways. It is largely unknown how cellular structures or components spatiotemporally regulate LLPS and condensate formation. Here we reveal that cytoskeletal dynamics can control the condensation of p62 bodies comprising the autophagic adaptor p62/SQSTM1 and poly-ubiquitinated cargos. Branched actin networks are associated with p62 bodies and are required for their condensation. Myosin 1D, a branched actin-associated motor protein, drives coalescence of small nanoscale p62 bodies into large micron-scale condensates along the branched actin network. Impairment of actin cytoskeletal networks compromises the condensation of p62 bodies and retards substrate degradation by autophagy in both cellular models and Myosin 1D knockout mice. Coupling of LLPS scaffold to cytoskeleton systems may represent a general mechanism by which cells exert spatiotemporal control over phase condensation processes.

Struggle for survival: new insights into NELF condensation for adaptive transcriptional reprogramming.

In this issue of Molecular Cell, Rawat et al. (2021) characterize novel stress-induced condensates of the negative elongation factor (NELF) as the nuclear counterparts of cytosolic stress granules. This provides a new perspective on transcription repression orchestrated by phase separation.

Acetylation of ezrin regulates membrane-cytoskeleton interaction underlying CCL18-elicited cell migration.

Ezrin, a membrane-cytoskeleton linker protein, plays an essential role in cell polarity establishment, cell migration, and division. Recent studies show that ezrin phosphorylation regulates breast cancer metastasis by promoting cancer cell survivor and promotes intrahepatic metastasis via cell migration. However, it was less characterized whether there are additional post-translational modifications and/or post-translational crosstalks on ezrin underlying context-dependent breast cancer cell migration and invasion. Here we show that ezrin is acetylated by p300/CBP-associated factor (PCAF) in breast cancer cells in response to CCL18 stimulation. Ezrin physically interacts with PCAF and is a cognate substrate of PCAF. The acetylation site of ezrin was mapped by mass spectrometric analyses, and dynamic acetylation of ezrin is essential for CCL18-induced breast cancer cell migration and invasion. Mechanistically, the acetylation reduced the lipid-binding activity of ezrin to ensure a robust and dynamic cycling between the plasma membrane and cytosol in response to CCL18 stimulation. Biochemical analyses show that ezrin acetylation prevents the phosphorylation of Thr567. Using atomic force microscopic measurements, our study revealed that acetylation of ezrin induced its unfolding into a dominant structure, which prevents ezrin phosphorylation at Thr567. Thus, these results present a previously undefined mechanism by which CCL18-elicited crosstalks between the acetylation and phosphorylation on ezrin control breast cancer cell migration and invasion. This suggests that targeting PCAF signaling could be a potential therapeutic strategy for combating hyperactive ezrin-driven cancer progression.

Holliday junction recognition protein interacts with and specifies the centromeric assembly of CENP-T.

The centromere is an evolutionarily conserved eukaryotic protein machinery essential for precision segregation of the parental genome into two daughter cells during mitosis. Centromere protein A (CENP-A) organizes the functional centromere via a constitutive centromere-associated network composing the CENP-T complex. However, how CENP-T assembles onto the centromere remains elusive. Here we show that CENP-T binds directly to Holliday junction recognition protein (HJURP), an evolutionarily conserved chaperone involved in loading CENP-A. The binding interface of HJURP was mapped to the C terminus of CENP-T. Depletion of HJURP by CRISPR-elicited knockout minimized recruitment of CENP-T to the centromere, indicating the importance of HJURP in CEPN-T loading. Our immunofluorescence analyses indicate that HJURP recruits CENP-T to the centromere in S/G2 phase during the cell division cycle. Significantly, the HJURP binding-deficient mutant CENP-T6L failed to locate to the centromere. Importantly, CENP-T insufficiency resulted in chromosome misalignment, in particular chromosomes 15 and 18. Taken together, these data define a novel molecular mechanism underlying the assembly of CENP-T onto the centromere by a temporally regulated HJURP-CENP-T interaction.