The EMBO Journal

Coronaviruses hijack host membranes to assemble ER-derived double-membrane vesicles (DMVs) that shield viral RNA replication from the cell intrinsic surveillance. Although DMVs morphologically resemble autophagosomes, whether and how autophagy factors actively support their biogenesis has remained elusive. Here, we identify a non-canonical requirement for the autophagy protein LC3C in {beta}-coronavirus replication. Loss of LC3s impaired viral RNA replication, whereas genetic ablation of ATG7 did not, indicating that canonical ATG7-dependent lipidation is dispensable in this context. Reconstitution experiments showed that only LC3C substantially restored replication in LC3-deficient cells and that LC3C phospho-mutants, differing in accessibility to ATG4-mediated processing, displayed distinct proviral activities. Additionally, ATG4D, the main protease responsible for maintaining the LC3 non-lipidated pool, is selectively required for viral replication. Both ATG4D and LC3s depletion triggers formation of aberrant DMV-like structures and potently suppresses SARS-CoV-2 replication. Ultrastructural analysis of nsp3-nsp4-induced membranes showed that depletion of LC3s or ATG4 proteases altered DMV abundance and morphology, supporting a role for the LC3C-ATG4D axis in replication organelle biogenesis. These data establish that {beta}-coronaviruses repurpose ATG4D-driven LC3C de-lipidation for non-canonical LC3 recruitment to replication organelles, identifying the lipidation state of LC3 as a molecular determinant of replication organelle biogenesis and efficient viral replication. HighlightsThe manuscript shows that {beta}-coronavirus replication depends on LC3 proteins and particularly on LC3C in reconstitution experiments, that this dependency is independent of ATG7-mediated lipidation, and that ATG4D promotes efficient replication and replication organelle morphology. Together, the data support a model in which a non-canonical LC3C-ATG4D pathway contributes to DMV biogenesis and viral RNA replication.

Huntingtin-associated protein 40 (HAP40) is an obligate structural subunit of huntingtin (HTT) and is rapidly degraded when unbound, yet has been conserved across eukaryotes for over a billion years. Combining interactomics, quantitative respirometry, and transcriptomics, we show that the HTT-HAP40 complex functions as a bidirectional stoichiometric rheostat: unbuffered apo-HAP40 activates the Integrated Stress Response via ATF4 and DDIT3/CHOP, whereas unbuffered apo-HTT reciprocally drives cholesterol and fatty-acid biosynthesis through SREBF1/2. We identify the ER-mitochondria tether RMDN3 (PTPIP51) as a key HAP40 interactor, placing mitochondria-associated ER membranes (MAMs) at the rheostats convergence point, and demonstrate that HAP40 depletion specifically impairs respiratory complexes II/IV. Loss of rheostat balance reproduces transcriptional signatures of Huntingtons disease patient tissues, supporting a "dual failure" model in which collapse of stoichiometric buffering -- rather than aggregation toxicity alone -- drives pathogenesis. To our knowledge, this is the first obligate complex in which both unbound partners carry out distinct essential functions, defining stoichiometric buffering as a generalizable regulatory principle that couples complex assembly to metabolic and stress-response control across eukaryotes.

The ubiquitin-proteasome system is the major pathway for selective protein degradation in eukaryotic cells. Some proteins are degraded by the proteasome without ubiquitination, but the prevalence and underlying mechanisms remain poorly understood. Here, we use pulsed-SILAC proteomics to systematically identify proteins undergoing ubiquitin-independent proteasomal degradation (UbInPD). We identify the paralogous proteasome activation factors ZFAND5 and ZFAND6 (ZFAND5/6) and show that ZFAND5 contains a ubiquitin-independent degron that can both promote its rapid turnover and allosterically activate the proteasome. These findings support a model in which proteasome activation and activator degradation are coupled through a ubiquitin-independent feedback mechanism. We further show that ZFAND5/6, together with the scaffold protein p62, restrain NF-{kappa}B activation in the TLR4 pathway. We link this regulation to the degradation of UBCH5c, an abundant E2 ubiquitin-conjugating enzyme that can initiate or prime ubiquitin chain synthesis. Our findings expand the known landscape of UbInPD and reveal unexpected links between proteasome activation, E2 enzyme regulation, and inflammatory signalling.

Molecular chaperones are known for their role in preventing protein aggregation and assisting proteins in reaching their structurally functional state. DNAJB6, a J-domain protein that partners with Hsp70s and nucleotide exchange factors, is very potent in preventing amyloid formation of proteins with large intrinsically disordered regions (IDRs), including several disease-associated proteins. Complementary to this, we recently demonstrated a role for DNAJB6 in surveilling FG-Nucleoporins (FG-Nups) phase transitions and highlighted its role in nuclear pore complex assembly. We expand on this by showing that this activity of phase state surveillance is directed to several FG-Nups and shared with the closely related DNAJB2 and DNAJB8. We demonstrate that the surveillance mechanism of DNAJB6 is encoded in an unusually highly conserved IDR that promotes the formation of stable, gel-like assemblies of the chaperone itself. These assemblies likely provide a stable environment that can outcompete stable homotypic FG-Nup interactions and instead favors multivalent heterotypic chaperone:FG-Nup interactions. The evolutionary conservation of the DNAJB6-IDR, mutant analyses from both experimental in vitro and in cell data, and multiscale molecular dynamics simulations suggest that the sequence space for encoding stable gel-like assemblies is narrow and optimized to avoid self-aggregation while providing potent anti-amyloidogenic capacity.

Human LonP1 is an ATP-dependent mitochondrial protease that degrades damaged or redundant proteins. Indiscriminate proteolysis by LonP1 is limited through tight coordination of substrate recognition, unfolding, translocation and catalytic cleavage, yet the role of ATP hydrolysis in these individual steps remains unclear. Here, we show that LonP1 binds substrates and cleaves peptide bonds without ATP hydrolysis, whereas degradation of folded proteins strictly depends on ATP-driven unfolding and translocation. Initial substrate binding opens a closed ADP-bound resting state, enabling nucleotide exchange and stimulating ATPase activity. The opening also increases accessibility of the proteolytic chamber, modestly enhancing peptidase activity. Maximal peptidase activity is observed in a transition-state mimic stabilised by ADP{middle dot}AlF, in which substrate is engaged within the translocation channel. Cryo-EM analysis reveals that in this state the proteolytic active sites are no longer occluded, linking ATP-driven substrate translocation to full proteolytic activation. Together, these findings reveal how LonP1 prevents indiscriminate proteolysis during substrate selection by ensuring that efficient proteolysis occurs only in substrate-translocating states. Model of the conformational landscape and functional cycle of LonP1Schematic overview of LonP1 states and their inter-conversion. State transitions are modulated by substrate, nucleotide occupancy, temperature, and inhibitors. Key distinguishing features include the presence or absence of the lateral gap, nucleotide state, substrate engagement within the A-tunnel, and the handedness of the ATPase (A) domains. Additional indicators include the compactness of the proteolytic (P) domain and the presence of substrate density within the N-terminal (N) domain or at the coiled-coil domain (CCD) as well as the position of a loop within the catalytic centre. The depicted cryo-EM structures represent a model of a continuous conformational landscape and correspond to the closest matching biological states and positions within the reaction cycle, but may also capture transient intermediates or conformations stabilised by experimental conditions. The shown atomic models correspond to the states highlighted in larger font (R-state: PDB 7NGL; P1-state: PDB 7NFY; P2-state: PDB 7NGC; closed LonP1-ADP-substrate: PDB 9CC1). O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/733973v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@16e0491org.highwire.dtl.DTLVardef@1ee02b1org.highwire.dtl.DTLVardef@f2b47aorg.highwire.dtl.DTLVardef@26f6b2_HPS_FORMAT_FIGEXP M_FIG C_FIG

MTCH2 (mitochondrial carrier homolog 2) is a noncanonical member of the solute carrier family with five transmembrane (TM) helices, localized to the mitochondrial outer membrane. MTCH2's atypical topology creates a membrane-accessible hydrophilic groove, predicted to be necessary for its protein insertase and lipid scramblase activities. MTCH2 is linked to lipid metabolism and obesity and is required for starvation-induced mitochondrial hyperfusion. Here, we show that MTCH2 is a stable component of a complex containing the Armadillo (ARM) repeat-containing protein, ARMC1, and the DnaJ/Hsp40 chaperone protein, DNAJC11. Protein crosslinking, protein structural modeling, and molecular dynamics simulations demonstrate that the ARMC1 alpha-helical C-terminal domain (CTD) inserts into and stably interacts with the MTCH2 hydrophilic groove and blocks its lipid scramblase activity. We observe that starvation-induced mitochondrial fatty acid oxidation (FAO) is negatively regulated by the ARMC1-MTCH2 interaction. In ARMC1-deficient cells, FAO is stimulated compared to wild-type cells and lipid droplet abundance is significantly reduced. The altered lipid phenotype of ARMC1-/- cells is strictly dependent on MTCH2 and is reversed by ARMC1 expression in a manner dependent on its CTD. Beyond this metabolic axis, we also identify a function for ARMC1 in regulating lysosomal distribution and autophagic flux that is independent of its CTD and interaction with MTCH2. Thus, our data support a model in which the MTCH2-ARMC1 interaction functions as a metabolic switch during starvation to regulate the balance between fat storage and fat burning.

The recruitment and activation of abscission machinery following mitosis is tightly regulated in time and space, yet the mechanisms controlling this process are poorly understood. We find that RNA localization and local translation at the midbody regulates when and where abscission-regulating proteins are expressed. The 3'UTR of NET1 mRNA contains an element that is necessary and sufficient for RNA targeting to the midbody. Mislocalization of NET1 mRNA results in a loss of NET1 protein, a Rho family GEF, throughout the intercellular bridge as well as slower cell proliferation and delayed abscission. This leads to a loss of Arp2/3 at the abscission site and is dependent upon NET1 binding to Rho family GTPases. These findings establish midbody RNA localization and local translation as a key layer of regulation over abscission timing and identify a role for NET1 as a regulator of Arp2/3-mediated branched actin accumulation at the abscission site.

Eukaryotic cells contain multiple biochemically distinct actin filament networks, which enable versatile functions of actin in a range of cellular processes. Yet, the mechanisms by which specific actin filament networks are assembled in a common cytoplasm remain elusive. Here, we investigated how functionally distinct actin nanoscale layers, specified by -actininand tropomyosin isoforms, Tpm1.6 and Tpm3.2, are assembled at focal adhesions. By combining genetic perturbations with mitochondrial-targeting of actin polymerases, we discovered that DAAM1 formin assembles Tpm3.2-actin filaments, whereas Ena/VASP family proteins polymerize -actinin cross-linked actin filament bundles at focal adhesions. Consequently, loss of DAAM1 dampened Tpm3.2 protein levels and impaired focal adhesion disassembly, thus phenocopying Tpm3.2-deficient cells. In contrast, Ena/VASP depletion led to defective focal adhesion maturation and loss of -actinin from focal adhesions. More broadly, our study highlights specific roles for formin and Ena/VASP family proteins in assembling biochemically and functionally distinct linear actin filament arrays in cells.

Microtubule dynamic instability, driven by GTP turnover, allows microtubules in cells to re-organise themselves adaptively. In some models of dynamic instability, GTP-tubulin is selectively captured at the tips of microtubules. In others, GTP- and GDP-tubulin are both captured, but GTP-tubulin is selectively retained. To investigate, we mutated the interprotofilament interface in human 1b{beta}3 and 1b{beta}4b tubulins, whose sequences diverge markedly in this region. We find that transplanting the 1b{beta}3 M-loop or its binding pocket into 1b{beta}4b tubulin creates tubulins that assemble in 1 mM GDP. In co-assembly experiments in GTP, such hyper-assembler mutants can recruit hypo-assembler mutants into a mosaic lattice, under conditions in which the hypo-assembler alone does not polymerise. We propose that GTP- and GDP-tubulins are captured equivalently at the tips of microtubules, but then differentially retained, based on their differing abilities to form stable interprotofilament bonds. This biased retention mechanism allows mosaic lattices to be built and dynamic instability to be tuned.

Tioxazafen is an effective nematicide whose commercialization was halted because handlers reportedly developed rashes after working with seeds coated with a tioxazafen-laced cocktail. Here, we show that tioxazafen is bioactivated into toxic products by nematode and human cytochrome P450s. Through systematic analyses, we found that bioactivated tioxazafen disrupts proteasome function, leading to the accumulation of the NRF1 transcription factor ortholog SKN-1A in the nematode C. elegans, and a bounce-back transcriptional up-regulation of proteasome components. Genetic upregulation of the C. elegans proteasome supresses tioxazafens lethality, indicating that proteotoxicity is a key contributor to death. A survey of human P450s revealed that skin-expressed CYP1A1 toxifies tioxazafen and may account for the reaction to tioxazafen-coated seeds. We also found that rabbit CYP1A1 fails to bioactivate tioxazafen, which may explain the pre-market failure to detect robust adverse skin reactions. Our work highlights vulnerabilities in pre-market toxicological assays and a provides potential solution to prospectively identifying P450 toxication events. One-Sentence SummaryTioxazafen is bioactivated by cytochrome P450s into a proteasome disruptor.

KRAS mutations drive some of the most lethal carcinomas, and genomic and inducible systems have established many of the cellular and tissue-level consequences. However, these approaches operate at the level of oncogene expression, allowing for cellular adaptation that masks the individual role of KRAS oncoprotein signaling. Here, we developed a reversible Opto-Chemical system to activate KRAS signaling by chemically translocating a cytosolic mutant KRASG12V G-domain to the plasma membrane upon light or small-molecule input. In MDCK cells, the G-domain plasma membrane recruitment activated downstream signaling and reduced collective migration. In mouse small Intestinal Organoids, G-domain recruitment promoted increased crypt size and number under Epidermal Growth Factor-deprived conditions. We further showed that the increased number of crypts depended on continuous KRASG12V signaling. Finally, under the same deprived conditions, localized activation in just one budding crypt promoted crypt formation compared to controls. This system decouples oncoprotein activity from oncogene expression, allowing to investigate the KRAS signaling contribution to early epithelial transformation.

The polarization of motile cilia requires that the centrioles, from which cilia are formed, display rotational asymmetry. This property is manifested in the presence of asymmetrically distributed appendages and relies on evolutionary conserved mechanisms. These mechanisms are also at play in cells that form primary cilia despite the lack of ciliary motility and asymmetric centriole appendages in this context. Here, we find that a complex consisting of CCDC61, KIAA1328 (K1328), and Centlein (CNTLN) contributes to the establishment of centriole rotational asymmetry. In cells with a primary cilium, this complex is required for assembling a linker that repositions the daughter centriole close to and orthogonal to the proximal end of the mother centriole/basal body. The CCDC61/K1328/CNTLN complex also triggers the asymmetric recruitment of pericentriolar matrix components around newly assembled centrioles, which likely facilitates the later attachment of the basal body-daughter centriole linker. Overall, our results establish that rotational asymmetry relies on the coordinated recruitment of asymmetric landmarks along centrioles and is necessary for positioning the centrioles in a configuration that is widely conserved in ciliated cells.

mRNAs can form stable structures that need to be resolved to facilitate translation. During translation initiation in mammals, the scanning 48S complex requires the helicase activity of DHX29 to unwind stable mRNA structures that cannot be resolved by eIF4A. Here, we show that the yeast DHX29 homolog, Ylr419w (Dhx29), has a similar function during translation on elongating 80S ribosomes. Cryo-EM analyses show that the Dhx29 helicase module is positioned at the mRNA entry channel to engage mRNA, while its double-stranded RNA-binding domain (dsRBD) senses hairpin-forming mRNA in the ribosomal A-site. By selective ribosome profiling, we observed that Dhx29 is associated with transcripts that form RNA structures, such as stable tetraloops. Dhx29 mutants with perturbed helicase activity enrich 80S with hairpins in the A-site, as well as ribosome collisions, while a mutant lacking the N-terminal dsRBD sensor domain loses the specificity for such ribosomes. We thus propose that Dhx29 functions in translation elongation by resolving structured mRNA formed in the ribosomal A-site through its 3'-5' helicase activity and pulling on the mRNA from its 3' end.

O-linked {beta}-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is the sole writer for intracellular O-GlcNAcylation. It catalyzes O-GlcNAcylation of thousands of protein substrates, but relatively less is known about the post-translational modifications that occur on OGT itself. Herein, we demonstrate that OGT is S-palmitoylated at Cys-472 and Cys-477, which is mediated by the S-acyltransferase Zinc Finger DHHC-Type Palmitoyl transferase 14 (zDHHC14) and removed by acyl protein thioesterase 2 (APT2). S-Palmitoylation stabilizes OGT by shunting it away from the lysosomal chaperone-mediated autophagy (CMA) pathway, as S-palmitoylation decreases the interaction between OGT and heat shock cognate 70 kDa protein (HSC70), the CMA chaperone. Via label-free quantitative mass spectrometry, we find that S- palmitoylation elevates the affinity between OGT and protein phosphatase 1 catalytic subunit gamma (PPP1CC), but not PPP1CB. We further demonstrate that S-palmitoylation of OGT augments binding with Yes-associated protein-1 (YAP), a protein that associates with PPP1CC, and subsequently enhances YAP O-GlcNAcylation. Our work unearths S-palmitoylation of OGT and CMA-mediated degradation of lysosomal OGT, the orchestration of which finetunes the activity of key OGT complexes, such as OGT-PPP1CC, and contributes to OGT substrate selectivity.

Rubicon is a negative regulator of autophagy and the endolysosomal network (ELN) and an antagonist of the class III phosphatidylinositol 3-kinase complex II (PI3KC3-C2). Inhibition of Rubicon is considered a potential means to therapeutically upregulate autophagy and the ELN to treat Parkinsons disease and other conditions characterized by autophagic and ELN dysfunction. Rubicon is specific for the UVRAG-containing PI3KC3-C2 over the purely autophagic ATG14- containing PI3KC3-C1 complex. Here, we determined the high-resolution cryo-electron microscopy structure of PI3KC3-C2 in complex with the PI3KC3-binding domain (PIKBD) of Rubicon and compared it to cryo-EM structures of unbound PI3KC3-C2 and PI3KC3-C1. Rubicon binds directly to PI3KC3-C2 only via the BARA domain of the BECN1 subunit, which is common to both C1 and C2. The selectivity of Rubicon for the PI3KC3-C2 complex over the PI3KC3-C1 complex is attributed to a conformation of the BECN1BARA domain induced by UVRAG, rather than to direct contact with UVRAG or direct antagonism by the ATG14 subunit of PI3KC3-C1. Targeted disruption of the Rubicon:PI3K3-C2 structural interface by site-directed mutations enhances mitophagic activity in human epithelial cells to levels comparable to those observed in Rubicon knockout (KO) cells. Similarly, disruption of the interaction in Rubicon-overexpressing hippocampal neurons restored lysosomal flux to wild-type levels. These data show that suppressing the function of PI3K3- C2 can fully account for the negative regulatory effects of Rubicon in the autophagy and ELN pathways. Significance StatementEndolysosome maturation and autophagosome-lysosome fusion require the production of phosphatidylinositol 3-phosphate (PI(3)P) by the class III phosphatidylinositol 3-kinase complex II (PI3KC3-C2). Rubicon is a key negative regulator of endolysosomes and autophagy that suppresses PI3KC3-C2 activity. Here, we reveal in atomistic detail how Rubicon selectively recognizes PI3KC3-C2. Disrupting the Rubicon-PI3KC3-C2 interaction restores mitophagy and enhances lysosomal activity to the same extent as Rubicon gene deletion, establishing that PI3KC3-C2 inhibition fully accounts for the biological regulatory effects of Rubicon in the autophagy and lysosome pathways.

Set promotes cohesion removal in mitosis by evicting phosphorylated Histone H1 and counteracting Sgo1. In addition, Set promotes chromosome alignment by counteracting Aurora B activation. The underlying molecular mechanisms through which Set performs these activities remain insufficiently characterized, but roles of Set as a Histone chaperone and PP2A inhibitor have been proposed. Building on our previous observations that Set promotes pericentromeric Cohesin removal in oocyte meiosis II, we generated an oocyte-specific conditional knock-out of Set to address its functions in meiosis. Similar to mitosis, Set depletion caused chromosome alignment and cohesion defects. We found that Set is required for accurate error correction by localizing Aurora B/C, and for efficient cleavage of the meiosis-specific Cohesin subunit Rec8 by Separase. Paired chromosomes and sister chromatids were often incompletely separated, likely a primary cause of missegregation. Set performed both its roles in a Sgo2-dependent manner, but, unexpectedly, independently of interaction with PP2A-B56. In line with a role of Set as a Histone chaperone, accumulation of phosphorylated Histone H1 in Set knock-out oocytes occurs concomitantly with reduction of oocyte-specific H1foo on chromosome arms, indicating that Set is required to create the optimal chromatin environment for efficient Rec8 cleavage by Separase in meiosis.

Circadian rhythms in mammals are generated by negative feedback loops, in which CLOCK and BMAL1 bind to E-box to activate transcription of Period (Per) and Cryptochrome (Cry) and the E-box-dependent transactivation is inhibited by PER and CRY proteins. Although the core transcriptional feedback loop of the circadian clock has been well defined, how this machinery interfaces with broader nuclear regulatory systems remains incompletely understood. Here, we identify nardilysin (NRDC), a metalloendopeptidase previously implicated in nuclear transcriptional regulation and metabolic homeostasis, as an unexpected modulator of the circadian clock. NRDC deficiency led to elevated PER2 protein levels in the liver and enhanced PER2 dynamics in cell-autonomous circadian oscillators, and was accompanied by a significant shortening of behavioral rhythms in mice. Biochemical analyses demonstrated that NRDC selectively associates with PER2 and CRY2 and antagonizes PER2-mediated repression of CLOCK-BMAL1-dependent transcription. Genome-wide chromatin immunoprecipitation analyses reveal that NRDC is enriched at promoter-proximal E-box-containing regions, frequently co-localizing with CLOCK binding sites. Together, these findings uncover a previously unrecognized link between circadian timing and protease-based nuclear regulation, positioning NRDC as a critical modulator of PER2 function and circadian period determination.

Bacterial spores owe their remarkable resistance properties to a multilayered coat, one of the most resilient and durable biological structures on Earth. Assembled at the surface of the outer forespore membrane, the coat comprises dozens of proteins organized into distinct layers. Its formation is initiated by SpoIVA, which is proposed to form a polymeric scaffold for the innermost coat layer. Although SpoIVA has been shown to polymerize into filaments in vitro, there is currently no evidence demonstrating the formation of such assemblies in vivo, and the mechanism underlying its oligomerization remains unresolved. In this study, cryo-focused ion beam milling combined with cryo-electron tomography of sporulating Bacillus subtilis cells reveals that the SpoIVA layer consists of polymers that form track-like structures radiating from the mother cell-proximal forespore pole and extending directionally toward the distal pole. Subtomogram averaging further sheds light on their organized architecture, harboring a straight orientation, uniform spacing, and embedding in the outer forespore membrane. These observations also define SpoIVA spatial orientation relative to the outer forespore membrane. Furthermore, AlphaFold3 predictions, combined with biophysical and functional assays, show that SpoIVA dimerizes through its central and C-terminal regions. We further show that dimerization promotes SpoIVA localization around the forespore but is dispensable for polymer formation, which relies on the ATPase domain. Altogether, these findings suggest a dual oligomerization mechanism, in which SpoIVA transitions from dimers to linear track-like polymers, and reveal that these assemblies play critical roles in coat assembly and spore development.

During spermiogenesis, early round spermatids differentiate into specialized spermatozoa through an extensive reshaping of the nucleus driven by coordinated cytoskeletal and chromatin-based mechanisms. In mammals, this process critically relies on the transient manchette, a microtubule-based structure that remodels the spermatid nucleus and serves a track to transport material required for flagellum assembly. However, the existence, organization, and molecular composition of such a structure in other vertebrates have remained poorly investigated. Here, we establish that an organized microtubule network is present in Xenopus spermatids and shares key architectural and molecular hallmarks of the mammalian manchette. We further uncover a large structural heterogeneity of spermatid microtubules, with variable protofilament numbers, skew angles, and lattice organizations. We reveal the presence of a Spaca9-Saxo2 internal scaffold in spermatid microtubules suggesting an internal reinforcement mechanism necessary for extensive nuclear reshaping and cytoplasm remodeling.

Angstrom-scale changes in microtubule (MT) lattice spacing regulate the selective recruitment of MT-associated proteins, yet how these structural states operate in cells remains poorly understood. Here, we show that MT lattice expansion, induced by protein-based expanders or microtubule-stabilizing agents such as Taxol and epothilone D, drives the relocalization of compact lattice-binding proteins, including tau, doublecortin (DCX), and the C1 domain-containing signaling protein GEF-H1, into highly curved MT-associated domains, whereas the compaction-inducing agent laulimalide suppresses this response. In contrast, the tumor suppressor RASSF1A preferentially associates with expanded lattice states, revealing differential lattice sensitivity among closely related C1 domain-containing proteins. These short, curved assemblies are enriched at MT intersections and discrete MT segments, revealing spatially heterogeneous lattice states within individual microtubules. At substoichiometric levels, compact lattice-binding proteins behave as both MT compactors and curvature sensors. Changes in osmotic pressure selectively promote dissociation of compact lattice-binding proteins, whereas expanded lattice-binding proteins remain largely unaffected. Using curved filament formation as an in-cellulo readout of compact lattice regions, we identify widespread lattice-state sensitivity across diverse MT-associated and signaling proteins. Finally, we show that these principles extend to neurons, where somatic, but not axonal, tau exhibits sensitivity to lattice expansion despite the expanded lattice architecture of distal axonal microtubules, suggesting additional neuron-specific regulation of lattice accessibility. Together, our findings identify the MT lattice as a dynamic mechanochemical platform whose nanoscale structural states spatially organize protein recruitment and signaling in cells and neurons.