Transcriptome analysis of ovary culture-induced embryogenesis in cucumber (Cucumis sativus L.)

Background. Ovary culture is a useful technique used to generate double haploid (DH) cucumber (Cucumis sativus L.) plants. However, cucumber ovary culture have a low rate of embryo induction and plant regeneration. Moreover, the cucumber embryogenesis mechanism remains unclear. In this study, we explored the molecular basis of cucumber embryogenesis in order to establish a foundation for a more efficient ovary culture method. Using transcriptome sequencing, we also investigated the differential expression of genes during the embryogenesis process. Methods. Cytological and morphological observations have divided cucumber ovary culture into three stages: early embryo development (T0), embryo morphogenesis (T1, T2, T3 and T4), and shoot formation (T5). We selected six key time points for transcriptome sequencing and analysis: T0 (the ovules were cultured for 0 d), T1 (the ovules were cultured for 2 d), T2 (the embryos were cultured for 10 d), T3 (the embryos were cultured for 20 d), T4 (the embryos were cultured for 30 d), and T5 (the shoots after 60 d culture). Results. We used cytology and morphology to observe the characteristics of the cucumber’s developmental transformation during embryogenesis and plant regeneration. The differentially expressed genes(DEGs) at developmental transition points were analyzed using transcriptome sequencing. In the early embryogenesis stage, the cells expanded, which was the signal for gametophytes to switch to the sporophyte development pathway. RNA-seq revealed that when compared to the fresh unpollinated ovaries, there were 3,468 up-regulated genes in the embryos, including hormone signal transduction genes, hormone response genes, and stress-induced genes. The reported embryogenesis-related genes BBM, HSP90 and AGL were also actively expressed during this stage. In the embryo morphogenesis stage (from cell division to cotyledon-embryo formation), 480 genes that functioned in protein complex binding, microtubule binding, tetrapyrrole binding, tubulin binding and other microtubule activities were continuously up-regulated during the T1, T2, T3 and T4 time points. This indicated that the cytoskeleton structure was continuously being built and maintained by the action of microtubule-binding proteins and enzyme modification. In the shoot formation stage, 1,383 genes were up-regulated that were mainly enriched in phenylpropanoid biosynthesis, plant hormone signal transduction, phenylalanine metabolism, and starch and sucrose metabolism. These up-regualted genes included six transcription factors that contained a B3 domain, nine genes in the AP2/ERF family, and two genes encoding WUS homologous domain proteins. Conclusions. Evaluation of molecular gynogenesis events may contribute to a better understanding of the molecular mechanism of cucumber ovarian culture.

ER proteins decipher the tubulin code to regulate organelle distribution

Organelles move along differentially modified microtubules to establish and maintain their proper distributions and functions1,2. However, how cells interpret these post-translational microtubule modification codes to selectively regulate organelle positioning remains largely unknown. The endoplasmic reticulum (ER) is an interconnected network of diverse morphologies that extends promiscuously throughout the cytoplasm3, forming abundant contacts with other organelles4. Dysregulation of endoplasmic reticulum morphology is tightly linked to neurologic disorders and cancer5,6. Here we demonstrate that three membrane-bound endoplasmic reticulum proteins preferentially interact with different microtubule populations, with CLIMP63 binding centrosome microtubules, kinectin (KTN1) binding perinuclear polyglutamylated microtubules, and p180 binding glutamylated microtubules. Knockout of these proteins or manipulation of microtubule populations and glutamylation status results in marked changes in endoplasmic reticulum positioning, leading to similar redistributions of other organelles. During nutrient starvation, cells modulate CLIMP63 protein levels and p180–microtubule binding to bidirectionally move endoplasmic reticulum and lysosomes for proper autophagic responses.

Long-Range Electrostatic Interactions Significantly Modulate the Affinity of Dynein for Microtubules

The dynein family of microtubule minus-end directed motor proteins drives diverse functions in eukaryotic cells, including cell division, intracellular transport, and flagellar beating. Motor protein processivity, which characterizes how far a motor walks before detaching from its filament, depends on the interaction between its microtubule-binding domain (MTBD) and the microtubule. Dynein's MTBD switches between high- and low-binding affinity states as it steps. Significant structural and functional data show that specific salt bridges within the MTBD and between the MTBD and the microtubule govern these affinity state shifts. However, recent computational work suggests that non-specific, long-range electrostatic interactions between the MTBD and the microtubule may also play a significant role in the processivity of dynein. To investigate this hypothesis, we mutated negatively charged amino acids remote from the dynein MTBD-microtubule-binding interface to neutral residues and measured the binding affinity using microscale thermophoresis and optical tweezers. We found a significant increase in the binding affinity of the mutated MTBDs for microtubules. Furthermore, we found that charge screening by free ions in solution differentially affected the binding and unbinding rates of MTBDs to microtubules. Together, these results demonstrate a significant role for long-range electrostatic interactions in regulating dynein-microtubule affinity. Moreover, these results provide insight into the principles that potentially underlie the biophysical differences between molecular motors with various processivities and protein-protein interactions more generally.

Dynamic interactions and Ca2+-binding modulate the holdase-type chaperone activity of S100B preventing tau aggregation and seeding

The microtubule-associated protein tau is implicated in the formation of oligomers and fibrillar aggregates that evade proteostasis control and spread from cell-to-cell. Tau pathology is accompanied by sustained neuroinflammation and, while the release of alarmin mediators aggravates disease at late stages, early inflammatory responses encompass protective functions. This is the case of the Ca2+-binding S100B protein, an astrocytic alarmin which is augmented in AD and which has been recently implicated as a proteostasis regulator, acting over amyloid β aggregation. Here we report the activity of S100B as a suppressor of tau aggregation and seeding, operating at sub-stoichiometric conditions. We show that S100B interacts with tau in living cells even in microtubule-destabilizing conditions. Structural analysis revealed that tau undergoes dynamic interactions with S100B, in a Ca2+-dependent manner, notably with the aggregation prone repeat segments at the microtubule binding regions. This interaction involves contacts of tau with a cleft formed at the interface of the S100B dimer. Kinetic and mechanistic analysis revealed that S100B inhibits the aggregation of both full-length tau and of the microtubule binding domain, and that this proceeds through effects over primary and secondary nucleation, as confirmed by seeding assays and direct observation of S100B binding to tau oligomers and fibrils. In agreement with a role as an extracellular chaperone and its accumulation near tau positive inclusions, we show that S100B blocks proteopathic tau seeding. Together, our findings establish tau as a client of the S100B chaperone, providing evidence for neuro-protective functions of this inflammatory mediator across different tauopathies.

Hedgehog-induced ciliary trafficking of kinesin-4 motor KIF7 requires intraflagellar transport but not KIF7’s microtubule binding

The kinesin-4 motor KIF7 is a conserved regulator of the Hedgehog signaling pathway. In vertebrates, Hedgehog signaling requires the primary cilium, and KIF7 and Gli transcription factors accumulate at the cilium tip in response to Hedgehog activation. Unlike conventional kinesins, KIF7 is an immotile kinesin and its mechanism of ciliary accumulation is unknown. We generated KIF7 variants with altered microtubule binding or motility. We demonstrate that microtubule binding of KIF7 is not required for the increase in KIF7 or Gli localization at the cilium tip in response to Hedgehog signaling. In addition, we show that the immotile behavior of KIF7 is required to prevent ciliary localization of Gli transcription factors in the absence of Hedgehog signaling. Using an engineered kinesin-2 motor that enables acute inhibition of intraflagellar transport (IFT), we demonstrate that kinesin-2 KIF3A/KIF3B/KAP mediates the translocation of KIF7 to the cilium tip in response to Hedgehog pathway activation. Together, these results suggest that KIF7’s role at the tip of the cilium is unrelated to its ability to bind to microtubules.

Microtubule dynamics influence the retrograde biased motility of kinesin-4 motor teams in neuronal dendrites

Microtubules establish the directionality of intracellular transport by kinesins and dynein through polarized assembly, but it remains unclear how directed transport occurs along microtubules organized with mixed polarity. We investigated the ability of the plus-end directed kinesin-4 motor KIF21B to navigate mixed polarity microtubules in mammalian dendrites. Reconstitution assays with recombinant KIF21B and engineered microtubule bundles or extracted neuronal cytoskeletons indicate that nucleotide-independent microtubule binding regions of KIF21B modulate microtubule dynamics and promote directional switching on antiparallel microtubules. Optogenetic recruitment of KIF21B to organelles in live neurons induces unidirectional transport in axons but bi-directional transport with a net retrograde bias in dendrites. Removal of the secondary microtubule binding regions of KIF21B or dampening of microtubule dynamics with low concentrations of nocodazole eliminates retrograde bias in live dendrites. Further exploration of the contribution of microtubule dynamics in dendrites to directionality revealed plus-end-out microtubules to be more dynamic than plus-end-in microtubules, with nocodazole preferentially stabilizing the plus-end-out population. We propose a model in which both nucleotide-sensitive and insensitive microtubule binding sites of KIF21B motors contribute to the search and selection of stable plus-end-in microtubules within the mixed polarity microtubule arrays characteristic of mammalian dendrites to achieve net retrograde movement of KIF21B-bound cargos. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

K-fiber bundles in the mitotic spindle are mechanically reinforced by Kif15

The mitotic spindle, a self-constructed microtubule-based machine, segregates chromosomes during cell division. In mammalian cells, microtubule bundles called kinetochore-fibers (k-fibers) connect chromosomes to the spindle poles. Chromosome segregation thus depends on the mechanical integrity of k-fibers. Here, we investigate the physical and molecular basis of k-fiber bundle cohesion. We detach k-fibers from poles by laser ablation-based cutting, thus revealing the contribution of pole-localized forces to k-fiber cohesion. We then measure the physical response of the remaining kinetochore-bound segments of the k-fibers. We observe that microtubules within ablated k-fibers often splay apart from their minus-ends. Furthermore, we find that minus-end clustering forces induced by ablation seem at least partially responsible for k-fiber splaying. We also investigate the role of the k-fiber-binding kinesin-12 Kif15. We find that pharmacological inhibition of Kif15-microtubule binding reduces the mechanical integrity of k-fibers. In contrast, inhibition of its motor activity but not its microtubule binding ability, i.e., locking Kif15 into a rigor state, does not greatly affect splaying. Altogether, the data suggest that forces holding k-fibers together are of similar magnitude to other spindle forces, and that Kif15, acting as a microtubule crosslinker, helps fortify and repair k-fibers. This feature of Kif15 may help support robust k-fiber function and prevent chromosome segregation errors. [Media: see text] [Media: see text] [Media: see text]

The Pathogenic R5L Mutation Disrupts Formation of Tau Complexes on the Microtubule by Altering Local N-Terminal Structure

The microtubule-associated protein (MAP) Tau is an intrinsically disordered protein (IDP) primarily expressed in axons, where it functions to regulate microtubule dynamics, modulate motor protein motility, and participate in signaling cascades. Tau misregulation and point mutations are linked to neurodegenerative diseases, including Progressive Supranuclear Palsy (PSP), Pick's Disease and Alzheimer's disease. Many disease-associated mutations in Tau occur in the C-terminal microtubule-binding domain of the protein. Effects of C-terminal mutations in Tau have led to the widely accepted disease-state theory that missense mutations in Tau reduce microtubule-binding affinity or increase Tau propensity to aggregate. Here, we investigate the effect of an N-terminal disease-associated mutation in Tau, R5L, on Tau-microtubule interactions using an in vitro reconstituted system. Contrary to the canonical disease-state theory, we determine the R5L mutation does not reduce Tau affinity for the microtubule using Total Internal Reflection Fluorescence (TIRF) Microscopy. Rather, the R5L mutation decreases the ability of Tau to form larger order complexes, or Tau patches, at high concentrations of Tau. Using Nuclear Magnetic Resonance (NMR), we show that the R5L mutation results in a local structural change that reduces interactions of the projection domain in the presence of microtubules. Altogether, these results challenge both the current paradigm of how mutations in Tau lead to disease and the role of the projection domain in modulating Tau behavior on the microtubule surface.