Nucleus

Disassembly of the nuclear lamina is essential for successful mitosis in eukaryotic cells, but the mechanism that triggers the lamina breakdown remains unclear. Using 3D fluorescence microscopy imaging combined with 3D curvature analysis, we found that microtubule-dependent invaginations trigger lamina remodelling and lamin B1 network disruption. Monte Carlo simulations provide mechanistic insight into how those invaginations induce a mechanical discontinuity between isotropic expansion and anisotropic shrinking forces at the rim of the nucleus, which correlated with lamin B1 depletion sites and the location of cytoplasmic protein influx within the nucleus. Our findings suggest that pre-mitotic modifications of the lamina function as a mechanical trigger to facilitate nuclear rupture and are thereby a key driver of mitotic entry, complementing established biochemical pathways.

The mechanical properties of the nucleus are critical for maintaining nuclear integrity and function. We previously showed that chromatin dominates short-extension mechanics whereas lamins provide long-extension strain stiffening. To distinguish the roles of lamin isoforms, micromanipulation nucleus force measurements were performed on isolated nuclei from lamin A/C (Lmna-/-) and lamin B1 (Lmnb1-/-) knockout mouse embryonic fibroblast cells. Lamin A/C knockout does not alter short-extension nuclear stiffness but is essential for strain stiffening at longer extensions. Oppositely, lamin B1 loss reduced short-extension stiffness due to facultative heterochromatin loss while long-extension strain stiffening was slightly increased. Loss of lamin A/C and B1 resulted in similar lamin-chromatin linkers effects as LBR did not change and LAP2{beta} decreased in both. A simulation model of a polymeric lamin shell with stiff lamin A/C and softer lamin B1 subunits can qualitatively recapitulate experimental measurements of lamin knockout cells. Lamin A/C knockout resulted in abnormal nuclear shape but not nuclear blebbing or rupture whereas lamin B1 knockout, similar to other perturbations that cause heterochromatin loss, resulted in increased nuclear blebbing and rupture. This work illuminates the distinct mechanical roles of lamin A/C and B1 in determining nuclear structure and integrity.

While bioelectricity is increasingly recognized as an important regulator of cell function and morphogenesis, the field has almost exclusively focused on plasma membrane states. Voltage across the inner nuclear membrane (INM) has been proposed as a potential regulator of nuclear function, but how it responds to extracellular ionic perturbations and whether it relates to chromatin organization remain unclear. Here, we targeted the ratiometric genetically encoded voltage indicator ASAP3-R3 to SUN2-associated nuclear membranes in intact NRK cells and combined INM voltage measurements with Gray-Level Co-Occurrence Matrix (GLCM)-based chromatin texture analysis. Reporter localization was confirmed by fluorescence imaging and electron microscopy, and functional validation in isolated nuclei showed that sodium-potassium pump inhibition produced INM depolarization consistent with Goldman-Hodgkin-Katz (GHK)-based prediction. We then used our validated construct to determine the response of Vnuc and chromatin texture to changing ionic conditions via two exposure methods, gradual (ramped) exposure or direct application. In intact cells, ramping different sets of ionic solutions of decreasing sodium/increasing potassium, decreasing sodium, increasing potassium, or decreasing chloride induced INM hyperpolarization and coordinated changes in chromatin texture, including increased contrast and entropy, reduced homogeneity, and reduced nuclear area. These effects were strongly path-dependent, with nuclear responses shaped by the history and order of ionic exposure: sodium and potassium responses emerged most clearly during ramping exposure, whereas reducing chloride by direct exposure showed a more pronounced response profile. Direct changes in sodium exposure produced limited electrical and chromatin-texture effects, while direct potassium exposure altered chromatin texture and nuclear area without significantly changing VNuc. Importantly, shifting baseline chromatin state in either direction, through Trichostatin-A (TSA)-induced chromatin relaxation or sodium azide/2-deoxy-D-glucose-induced compaction, blunted ion-associated Vnuc and chromatin responses across sodium, potassium, and chloride conditions. Together, these findings identify the nucleus as a dynamic, ion-responsive electro-structural system in which INM voltage and chromatin organization are functionally coupled, and in which both ionic trajectory and pre-existing chromatin state shape the magnitude of the nuclear response.

BackgroundLamins are major regulators of the spatial and functional organization of chromatin. Lamins at the nuclear periphery form the lamina that anchors heterochromatin to the nuclear envelope. A subpool of A-type lamins localizes in the nuclear interior, where they also bind to euchromatic genomic regions. A-type lamin properties and chromatin association are regulated by lamin-associated polypeptide 2alpha (LAP2). Here we systematically analyze, how LAP2 depletion affects chromatin organization, accessibility and gene expression on a genome-wide level. ResultsLAP2 depletion in mouse dermal fibroblasts positively and negatively affects chromatin accessibility and gene expression throughout the genome, which correlates with changes in chromatin association of A-type lamins and the nucleosomal remodeler proteins BRG1 and CHD4. In particular, A-type lamins bind to open chromatin regions close to BRG1 and CHD4 binding sites and deregulated genes, but do not directly accumulate on genes and BRG1 and CHD4-enriched sites. Unsupervised clustering of the datasets on LAP2-bound genomic regions confirms spreading of A-type lamins to active chromatin regions containing deregulated genes and an enrichment of chromatin remodelers on a subset of these genomic regions. ConclusionsLAP2 depletion in fibroblasts leads to a gross rearrangement of chromatin. Genome-wide chromatin reorganization is linked to spreading of A-type lamins to active chromatin regions and accompanied by a restriction of chromatin remodelers to a subset of active genomic regions. These changes correlate with changes in chromatin accessibility and gene expression throughout the genome, particularly in regions where lamin binding is gained in LAP2 knockout versus wildtype cells.

Splicing of pre-mRNA can result in multiple possible mRNA isoforms per gene due to alternative splicing. The frequency at which individual isoforms occur depends on the intrinsic splicing kinetics of the pre-mRNA as well as intracellular chemical conditions. Computational modeling can potentially provide a platform to rapidly assess how variations in intracellular and environmental conditions, for example differential levels of regulatory splicing proteins, affect kinetics and resulting mRNA isoforms. Overcoming the vast combinatoric possibilities of splicing, however, has remained a significant challenge in modeling its kinetics. Here we report the development of a stochastic kinetic model of splicing that is extensible to most protein-coding genes in the human genome. Our model allows for variations in site-specific reaction rates as well as the ability to introduce additional splicing factors. We experimentally validate the predictive capability of our computational model by exploring the spliced isoform ratio of a target gene (SRSF6) under normoxia and hypoxia. This work provides a resource for quantitative, computational analysis of pre-mRNA splicing, allowing for a rapid computational-experimental approach to assess biological hypotheses. SignificancemRNA splicing is a key step in human gene expression with deep impact on the molecular processes determining cell physiology and affecting development and disease. Computational models of splicing are highly attractive to understand life processes but so far have been limited in directly accounting for the chemistry of splicing and are not extensible to most of the human genome. We report here a model that overcomes both of these challenges, providing a computational benchtop to probe splicing kinetics for most genes in the human genome. This model has the capability to rapidly pose biological hypotheses for experimental validation. As a targeted demonstration, we explore the effects of the pathologically-relevant chemical condition hypoxia on the pre-mRNA of a single gene.

H1 histones are abundant nuclear proteins that bind to linker DNA at the entry and exit points of nucleosomes. Although individual H1 family members play partially redundant roles in chromatin organization, the discovery of disease-associated mutations in H1 genes has raised interest in their cell-type specific functions. Heterozygous, de novo frameshift mutations in H1-4 cause the neurodevelopmental disorder Rahman Syndrome, which is characterized by mild to severe intellectual disability along with other neurological and morphological features. H1.4 has mostly been studied in dividing cells, and its expression in the brain was poorly understood. Here we characterized the expression of H1f4 mRNA and H1.4 protein in the brains of male and female mice across postnatal development. Using an epitope-tagged H1f4 knockin mouse, we show that this linker histone is robustly expressed throughout the brain including in mature, post-mitotic neurons of adult mice. By chromatin immunoprecipitation, we observe that H1.4 binds broadly across the genome in neural progenitors and accumulates in heterochromatin over the course of neuronal maturation. Finally we show that developmental maturation of chromatin compaction in cerebellar granule neurons is disrupted in H1f2/H1f4 double knockout mice. These data raise the possibility that the neurological changes in Rahman Syndrome may arise from disrupted functions of histone H1.4 in neurons.

Mechanical cues influence cell behavior and fate and are frequently accompanied by changes in nuclear shape; however, how epithelial nuclei accommodate such deformations remains incompletely understood. Here, we investigated the formation and regulation of nuclear wrinkles (NWs), inward folds of the nuclear envelope, in human epithelial cells. Using quantitative confocal imaging in 2.5D spheroid cultures and controlled 2D monolayers, we found that NWs formed frequently in MCF10A cells but rarely in hTERT-RPE1 cells, indicating pronounced cell-type specificity. NW frequency increased with cell density and was tightly associated with coordinated geometric changes consistent with nuclear rounding. Disruption of F-actin organization, but not microtubules, robustly induced NW formation, and acute cell rounding triggered by trypsinization was sufficient to induce widespread wrinkling across multiple cell types. Live-cell imaging revealed that NWs are dynamic and reversible at low cell density but become stabilized under sustained confinement. NW formation occurred without detectable nuclear envelope rupture, DNA damage, or stress-associated histone phosphorylation. Quantitative analysis supports a passive geometric model in which redistribution of excess nuclear surface area accommodates nuclear shape remodeling, allowing epithelial nuclei to buffer mechanical constraints while preserving nuclear integrity.

The size of the nucleus scales with cell size, suggesting a universal scaling rule. Yet the biophysical determinants of nuclear size and the significance and consequences of altered nuclear-to-cell (N/C) ratios, which are observed across diverse pathological states and cell-fate transitions, remain poorly understood. Recent theoretical models propose that nuclear size arises from a balance of colloid osmotic pressures generated by macromolecules in the nucleoplasm and cytoplasm. Here we demonstrate that altering this osmotic balance through massive overexpression of an exogenous protein targeted to either the nucleoplasm or cytoplasm produces predictable changes in the N/C ratio in S. pombe. These quantitative perturbations show that nuclear size is set primarily by the number of proteins in the nucleus and cytoplasm, providing strong support for a pure osmotic pressure mechanism. Furthermore, cells with altered N/C ratios display tunable changes in nucleoplasmic crowding, nuclear condensate formation, nucleolar scaling and heterochromatin organization, establishing a causal link between nuclear size and gene regulatory processes. These findings reveal how cells exploit osmotic forces to set organelle dimensions, with broad implications for understanding how nuclear size shapes gene expression and cell identity in health and disease.

BackgroundAcrodysostosis type 2 (ACRDYS2) is a rare autosomal dominant disease characterized by skeletal defects and cognitive deficit, with clinical symptoms observed in multiple other tissues including the skin. It is caused by mutations in a phosphodiesterase, PDE4D, a key regulator of cAMP/PKA (cyclic adenosine monophosphate / protein kinase A) signalling. Despite its well-defined genetic causes, the molecular mechanisms underlying the disease remain poorly understood, with studies based largely on engineered cellular models reaching conflicting interpretations. MethodsTo investigate how endogenous dynamics are affected by PDE4D mutations in unmanipulated cells, we studied PDE4D transcript and protein expression, activity and downstream signalling in native dermal fibroblast from ACRDYS2 patients and healthy controls. ResultsSignificant reduction in total PDE4D expression in patient cells was observed both at the transcript and protein level, with marked decreases in the long isoforms PDE4D4 and PDE4D7; a reduction in PDE4D9 mRNA was also observed. PDE4D enzymatic activity was reduced in ACRDYS2 fibroblasts, though total PDE activity was largely preserved. Reduced PDE4D expression was associated with an increase in the phosphorylated form of the cAMP-responsive transcription factor CREB and elevated PRKAR1A (PKA type 1 regulatory subunit alpha) transcript levels, suggesting altered downstream signalling. Interestingly, expression of the related phosphodiesterase family member PDE4B was increased, consistent with a compensatory response to reduced PDE4D function. ConclusionsThis is the first study demonstrating reduced PDE4D expression and isoform-specific dysregulation in native ACRDYS2 cells. Together, our results support a model in which reduction in PDE4D activity and compensatory changes in other PDE4 family members contribute to the molecular pathology of ACRDYS2, providing new insights into the molecular mechanisms underlying this disorder.

Yes-associated protein (YAP), a transcriptional coactivator, plays key roles in cell growth, proliferation and apoptosis, and its levels are frequently dysregulated in cancers. YAP levels in the nucleus are highly sensitive to nuclear mechanical cues, and such cues are also parallelly emerging to be a key modulator of DNA Damage Responses (DDR). However, whether DNA-damage can induce mechanical changes that regulate downstream events such as YAP localization and that in turn feeds back onto DDR activation, remains unknown. In this study, we report that YAP translocates in a nuclear mechanics-dependent manner upon induction of Double Strand Breaks (DSBs). This translocation is not a mere epiphenomenon, and we find that: first, global nuclear eviction of YAP enhances DDR signaling; second, local enrichment of YAP at DNA damage sites promotes recruitment of DNA repair proteins previously identified as potential interactors of YAP or its partner TEAD1. Together, these findings indicate that YAP is not only a transcriptional coactivator, but also plays an under-appreciated role in regulating DDR.

HP1 proteins are highly evolutionarily conserved chromatin-associated factors known to play essential roles in genome stability and nuclear organization. In mammals, three HP1 isoforms, HP1, HP1{beta} and HP1{gamma}, have been described, but their individual functions remain incompletely characterized. Here, we inactivated HP1 or HP1{beta} in different cell lines and quantified chromosomal breaks on metaphase spreads in the presence or absence of aphidicolin-induced replication stress. Loss of HP1, but not of HP1{beta}, led to a significant increase of chromosomal breaks on chromosome arms and within pericentromeric heterochromatin under these conditions. Mechanistically, loss of HP1 was associated with a reduction in replication fork velocity, suggesting that HP1 deficiency induces a replication stress that sensitizes specific genomic loci to replication perturbation. Consistent with this, HP1 loss was associated with a moderate but consistent increase in {gamma}H2AX and 53BP1 foci, an increased occurrence of DNA synthesis during mitosis, and enhanced recruitment of FANCD2, all recognized as hallmarks of common fragile site (CFS) expression. In addition, rescue experiments using a chromodomain mutant HP1 (V22M) unable to bind H3K9me3 indicated that HP1 protective function over these specific foci did not require its interaction with this histone mark. Altogether, these data indicate that, independently of its binding to H3K9me3, HP1 stabilizes specific genomic regions that behave as HP1-dependent fragile sites, at least in part by regulating replication fork progression, limiting mitotic DNA synthesis possibly by competing with FANCD2 for chromatin access at these regions.

Human nucleoli are multivalent, involving contributions from up to ten NOR-bearing acrocentric chromosome p-arms. Precision mega-base scale chromosome engineering defines the requirements for this major genome organisational event. NOR deletions reveal that p-arm nucleolar association is rDNA independent. Deletion of all NOR-distal or proximal sequences individually have only a marginal effect on nucleolar association. Finally, deletion of an entire p-arm, while leaving centromere function intact, destroys the nucleolar association potential of that acrocentric. We propose that formation of multivalent nucleoli is not a nucleolar fusion event per se; rather it is driven by the surrounding chromosomal context of NORs.

THAP9 is a transposable element-derived gene which encodes a protein that is homologous to the active Drosophila P-element transposase (DmTNP). Both THAP9 and DmTNP possess a C-terminal domain (CTD) which is functionally uncharacterized. Sequence and structural analysis suggest that the THAP9-CTD has a novel fold which is only found in THAP9 homologs. To explore the evolutionary history and characteristics of this novel domain, exhaustive phylogenetic analysis (using MSA, structure prediction, MSTA-based clustering) was performed. THAP9-CTD homologs were more widely distributed throughout the animal kingdom in comparison to DmTNP-CTD homologs which were restricted to arthropods. Moreover, the THAP9-CTD homologs were more conserved, especially among mammals and birds and their average length increased in a class-specific manner. Comparison with the DmTNP-CTD homologs demonstrates that although their respective CTDs may have evolved independently, they both surprisingly share similar secondary structure elements consisting of three conserved helical regions made of hydrophobic residues that are predicted to make up a conserved core. The role of the respective CTDs were further investigated by creating truncation mutants lacking the CTD. Interestingly both THAP9 and DmTNP truncation mutants are still capable of DNA excision and integration suggesting that their respective CTDs are not essential for DNA transposition. Moreover, CTD truncation favours DNA integration in THAP9: this suggests that CTD acquisition during evolution may have led to THAP9 domestication as observed in other transposable element-derived genes like Rag1 and piggybac, which have similar terminal regulatory domains.

Kinase fusion proteins (KFPs) have emerged as an important group of immune receptors encoded by plant resistance genes. Here, we report a new type of gene pair that controls resistance of wheat to the blast fungus, Pyricularia oryzae. We cloned a fungal gene involved in avirulence of P. oryzae pathotype Eleusine on wheat and designated it PWT8. We also identified its corresponding resistance gene in wheat, and tentatively named it Rwt8. This resistance gene was located at the same locus as previously identified resistance genes Rwt3 and Rwt6. Molecular cloning revealed that Rwt3, Rwt6, and Rwt8 were the same gene consisting of an identical gene pair, one encoding an NLR and the other encoding a mixed lineage kinase-like (MLKL) protein. These two genes were closely linked in a head-to-head orientation and behaved as a single gene. This gene pair recognized three AVR genes, PWT3, PWT6, and PWT8, and was designated Rwt3.6.8. The distribution of Rwt3.6.8 in common wheat landraces suggested that the gene pair may have been a factor which the D genome provided to the genus Triticum to broaden its adaptability to various environments in the world, especially in Asia and Africa.

Arabidopsis Leucine-Rich Repeat-Malectin Receptor Kinase (LRR-MAL RK) genes have been previously implicated in the early stages of pollen-pistil interactions to support compatible pollen. One member, Receptor Kinase in Flowers 1 (RKF1), has been associated with roles in the stigma to support pollen hydration as well as pollen tube growth. To better understand the function of RKF1 in these processes, a yeast two-hybrid screen was conducted with the RKF1 cytosolic kinase domain. Two positive interactors identified from this screen were the Group VII Ethylene Response Factors (ERFVIIs), RELATED TO APETALA 2.12 (RAP2.12) and RAP2.3. Their putative roles in pollen-pistil interactions were investigated using the quintuple erfvii mutant, and novel pistil-mediated pollen tube callose deposition phenotypes were uncovered during the pollen tube growth stage. Loss of seven LRR-MAL RKs including RKF1 in the pistil was previously found to cause an unusual phenotype where shorter callose plugs were deposited in wildtype pollen tubes compared to that seen in wildtype Col-0 pistils. Contrary to this, wildtype pollen tubes growing through the quintuple erfvii mutant pistil deposited callose plugs that were more elongated than that seen in wildtype Col-0 pistils. Further analyses with the proteolysis 6 (prt6) mutant and RAP2.12 rescue constructs were consistent with these phenotypes providing support that RKF1 is a negative regulator of RAP2.12 and RAP2.3 in the pistil during pollen tube growth.

The rejection of self-incompatible (SI) Brassica pollen is mediated by three signaling branches that function in parallel in the stigma. The recognition of SI pollen by the stigma S-Receptor Kinase (SRK) results in activation of the ARM-Repeat-Containing 1 E3 ubiquitin ligase (ARC1) which mediates the degradation of compatibility factors, the FERONIA (FER) and ANJEA (ANJ) receptor kinases that induces ROS accumulation to inhibitory levels and the M Locus Protein Kinase (MLPK) which may also be connected to ROS production. Arabidopsis self-incompatibility is regulated by SRK as well, but the signaling events downstream of SRK following SI pollen perception are less well-understood. In this study, we evaluated the requirements of FER, ANJ and HERCULES RECEPTOR KINASE 1 (HERK1) for SI pollen rejection in the transgenic Arabidopsis thaliana SI-Col-0{psi} srka-1 line. The{psi} srka-1 T-DNA disrupting the expression of the endogenous{psi} SRKA gene was crossed into SI-Col-0 to prevent any potential SRK transgene silencing. T-DNA mutants for FER and ANJ/HERK1 were then crossed into the SI-Col-0{psi} srka-1 line. Using standard assays for pollen-stigma interactions, the SI phenotypes were assessed for the SI-Col-0 fer, SI-Col-0 anj-1 and SI-Col-0 anj-1 herk1-1 lines. Our results presented here indicated that FER and ANJ are not required in the stigma for Arabidopsis SI pollen rejection, further providing evidence for a divergence in the SI downstream signaling pathway in Arabidopsis.

Telomeres are nucleoprotein structures that protect chromosome ends and are maintained by the Telomerase Reverse Transcriptase (TERT) protein that uses a noncoding Telomerase RNA (TR) as a template. In monkeyflowers, Mimulus lewisii had an ancient TR gene duplication, synthesizing an evolutionarily atypical sequence heterogeneous telomere. How TERT interacts with both TR paralogs during telomere maintenance is unknown and answers can shed novel insights underlying telomere function. Using new genome assemblies we discovered TERT is rapidly evolving in lineages sharing the TR duplication. We investigated the functional consequences arising from the rapid evolution, first by using yeast three-hybrid and testing the physical binding between conspecific and heterospecific TERT-TR combinations. Results showed TERT binds both ancestral (TR1) and derived (TR2) TR paralogs in M. lewisii, but not in species without a functioning TR2. We located the region of TR binding to amino acids near the KRxR motif. We then combined next-generation sequencing with Telomeric Repeat Amplification Protocol and discovered M. lewisii had high telomerase activity. Comparative transcriptomics indicated no strong evidence of expression divergence in telomere maintenance genes for M. lewisii, suggesting rapid evolution shaped TERT protein sequence. In vivo activity of M. lewisii telomerase was investigated by analyzing F1 telomeres generated by crossing M. lewisii and M. verbenaceus, which doesnt have a functioning TR2. Results showed M. verbenaceus chromosome ends in the F1 had converted into M. lewisii telomeres, suggesting dominance of the M. lewisii telomerase. We demonstrate TERT-TR coevolution can have significant consequences on the evolution of plant telomeres. Significance statementTelomeres protect chromosome ends and are maintained by the telomerase complex. We discovered the catalytic component of the telomerase (TERT) was rapidly evolving in monkeyflowers (Mimulus) and studied the molecular consequences. In M. lewisii, TERT evolved lineage-specific amino acids to bind two sequence divergent telomerase RNA paralogs. Telomerase activity assay showed M. lewisii synthesized more telomere repeats compared to its sister species without the TR duplication, and transcriptomics indicated this was not due to a change in telomere maintenance gene expression. Genetic experiments in interspecies hybrids showed M. lewisii telomerase could convert chromosome ends in sister species into M. lewisii-like telomeres suggesting functional dominance. We show rapid evolution of the telomerase can have significant effects on telomere evolution.

Nuclear organization is dynamic and originates from the fundamental subunit of chromatin, the nucleosome. Post-translational modification of nucleosomal histones, particularly within intrinsically disordered histone tail regions, provides a dynamic regulatory mechanism of accessibility for chromatin-templated processes. While the epigenomic impacts of lysine acetylation and serine phosphorylation in the histone H3 tail are well-known, how these charge-altering post-translational modifications (PTMs) alter nucleosomal tail conformational dynamics remains incompletely characterized. Given that the functional implications of these PTMs are, at least in part, a consequence of modified nucleosome conformation, systematically cataloging the impact of histone PTMs on nucleosome dynamics provides crucial insight into both baseline cellular activity and epigenetic dysregulation that occurs in disease. Previously, our lab demonstrated that arginine citrullination mimetics lead to regional increases in H3 tail dynamics within nucleosome core particles. Here, we performed nuclear magnetic resonance spin relaxation experiments to investigate the effects of lysine acetylation and serine phosphorylation on H3 tail picosecond-nanosecond (ps-ns) dynamics. Using lysine-to-glutamine and serine-to-glutamate mutations as acetyllysine and phosphoserine mimetics, respectively, we found that these PTMs increase ps-ns conformational dynamics regionally around the PTM site, with a position-dependent effect. Additionally, we show that the type of PTM influences the extent of these increases: in general, the effect of mimetics trends in the order of phosphorylation [&le;] acetylation < citrullination, suggesting a tunable method for altering histone tail dynamics. Taken together, these results illustrate the role of nucleosome conformational dynamics in conveying the effects of epigenomic PTMs, elucidating a mechanism of the histone language.

Changes in intracellular calcium ion (Ca{superscript 2}) concentrations generate characteristic signatures that are decoded by specialized Ca{superscript 2}-binding proteins (CaBP). Although substantial progress has been made in understanding cytosolic calcium signaling pathways, calcium signaling within organelles, particularly chloroplasts, remains poorly understood, partly because only a few EF-hand CaBP have been identified in organelles. Here, we describe a novel EF-hand protein of 18 kDa, that was found to be associated with the chloroplast envelope and peroxisomal membrane and was therefore named OEF18 (ORGANELLAR EF-HAND PROTEIN OF 18 kDa). OEF18 has a very unusual structure, containing an N-terminal myristoylation site, followed by one EF-hand in the N-terminus facing to the cytosol, and a transmembrane domain in the C-terminus. OEF18 membrane-targeting was found to be mediated by ANKYRIN REPEAT-CONTAINING PROTEIN 2A (AKR2A) via the C-terminal transmembrane domain of OEF18. Furthermore, the EF-hand in OEF18 bound Ca{superscript 2} at a physiological concentration that led to a large protein conformational change, inducing oligomerization of the N-terminal part. We found that oef18 mutants accumulated less jasmonic acid (JA) and its bioactive conjugate JA-Ile, likely causing a defect in the insect herbivore response. Wild-type OEF18 complemented the herbivory phenotype of oef18 mutants, whereas an EF-hand point mutant lacking Ca{superscript 2}-binding capacity failed to restore the wild-type response. Furthermore, OEF18 was required for resistance to salt stress in combination with dark-induced senescence. Together, these results establish OEF18 as a previously unrecognized organellar Ca{superscript 2} sensor that couples Ca{superscript 2} perception to JA-mediated defense and abiotic stress responses in plants.

Glucocorticoids are among the most widely prescribed drugs globally due to their potent anti-inflammatory and immunosuppressive actions. These effects are primarily mediated by the glucocorticoid receptor (GR), a ligand-activated transcription factor that translocates from the cytoplasm to the nucleus to regulate hundreds of genes. Although nuclear entry is a prerequisite for its genomic response, the mechanisms governing this process remain unresolved; specifically, whether the receptor translocates as a monomer or a dimer remains a subject of significant controversy. Here, we employed the pair correlation function (pCF) approach to quantify the nuclear translocation of single fluorescent GR molecules in live cells. This minimally invasive method identifies correlations between intensity fluctuations generated by molecules moving from the cytoplasm into the nucleus. Our results demonstrate that GRs quaternary structure and conformation modulate GR transport. While GR monomers rely exclusively on passive diffusion, GR dimers also utilize the microtubule-dynein machinery for active transport, proving that dimerization can precede nuclear import. Furthermore, the perinuclear vimentin network facilitates faster translocation by constraining actively transported dimers near nuclear pores. Collectively, our work reconciles contradicting reports regarding GR stoichiometry during import by demonstrating that both monomers and dimers translocate, albeit through distinct mechanisms. Importantly, these results reopen the door for a microtubule-dependent, heterocomplex-independent model of GR translocation, suggesting that the cytoskeleton is an integral, yet overlooked, component of the GR signaling pathway.