Amyloid deposition through endocytosis in vascular endothelial cells.

No mechanistic lead is known for establishing AL amyloid deposits in organs. We here report an electron microscopic (EM) analysis in a case of intestinal AL amyloidosis before initiating treatment for amyloidosis. The dense deposits of amyloid fibrils are concentrated around the small blood vessels in the submucosal area of intestinal tissue. Surprisingly, we observed endothelial cells (ECs) of blood vessels containing plenty of endocytotic (pinocytotic) and transcytotic vesicles at the luminal side and above the basement membrane, indicating the one-way active trafficking of either the immunoglobulin (Ig) light chain or preassembled amyloid fibrils from the luminal side of ECs to the extraluminal area of ECs. Immunoelectron microscopy displayed that the immuno-gold signals were observed in the vascular cavity and the subendothelial area of amyloid deposits. However, there is no sign of an Ig light chain in pinocytotic vesicles. Therefore, the intestinal ECs may actively pump out mainly the preassembled amyloid fibrils (not light chains) from the blood stream into the subendothelial area as a physiologic function.

Structural basis of Irgb6 inactivation by Toxoplasma gondii through the phosphorylation of switch I.

Irgb6 is a priming immune-related GTPase (IRG) that counteracts Toxoplasma gondii. It is known to be recruited to the low virulent type II T. gondii parasitophorous vacuole (PV), initiating cell-autonomous immunity. However, the molecular mechanism by which immunity-related GTPases become inactivated after the parasite infection remains obscure. Here, we found that Thr95 of Irgb6 is prominently phosphorylated in response to low virulent type II T. gondii infection. We observed that a phosphomimetic T95D mutation in Irgb6 impaired its localization to the PV and exhibited reduced GTPase activity in vitro. Structural analysis unveiled an atypical conformation of nucleotide-free Irgb6-T95D, resulting from a conformational change in the G-domain that allosterically modified the PV membrane-binding interface. In silico docking corroborated the disruption of the physiological membrane binding site. These findings provide novel insights into a T. gondii-induced allosteric inactivation mechanism of Irgb6.

CD47 promotes peripheral T cell survival by preventing dendritic cell-mediated T cell necroptosis.

Conventional dendritic cells (cDCs) are required for peripheral T cell homeostasis in lymphoid organs, but the molecular mechanism underlying this requirement has remained unclear. We here show that T cell-specific CD47-deficient (Cd47 ΔT) mice have a markedly reduced number of T cells in peripheral tissues. Direct interaction of CD47-deficient T cells with cDCs resulted in activation of the latter cells, which in turn induced necroptosis of the former cells. The deficiency and cell death of T cells in Cd47 ΔT mice required expression of its receptor signal regulatory protein α on cDCs. The development of CD4+ T helper cell-dependent contact hypersensitivity and inhibition of tumor growth by cytotoxic CD8+ T cells were both markedly impaired in Cd47 ΔT mice. CD47 on T cells thus likely prevents their necroptotic cell death initiated by cDCs and thereby promotes T cell survival and function.

TEAD1 trapping by the Q353R-Lamin A/C causes dilated cardiomyopathy.

Mutations in the LMNA gene encoding Lamin A and C (Lamin A/C), major components of the nuclear lamina, cause laminopathies including dilated cardiomyopathy (DCM), but the underlying molecular mechanisms have not been fully elucidated. Here, by leveraging single-cell RNA sequencing (RNA-seq), assay for transposase-accessible chromatin using sequencing (ATAC-seq), protein array, and electron microscopy analysis, we show that insufficient structural maturation of cardiomyocytes owing to trapping of transcription factor TEA domain transcription factor 1 (TEAD1) by mutant Lamin A/C at the nuclear membrane underlies the pathogenesis of Q353R-LMNA-related DCM. Inhibition of the Hippo pathway rescued the dysregulation of cardiac developmental genes by TEAD1 in LMNA mutant cardiomyocytes. Single-cell RNA-seq of cardiac tissues from patients with DCM with the LMNA mutation confirmed the dysregulated expression of TEAD1 target genes. Our results propose an intervention for transcriptional dysregulation as a potential treatment of LMNA-related DCM.

Structural model of microtubule dynamics inhibition by kinesin-4 from the crystal structure of KLP-12 -tubulin complex.

Kinesin superfamily proteins are microtubule-based molecular motors driven by the energy of ATP hydrolysis. Among them, the kinesin-4 family is a unique motor that inhibits microtubule dynamics. Although mutations of kinesin-4 cause several diseases, its molecular mechanism is unclear because of the difficulty of visualizing the high-resolution structure of kinesin-4 working at the microtubule plus-end. Here, we report that KLP-12, a C. elegans kinesin-4 ortholog of KIF21A and KIF21B, is essential for proper length control of C. elegans axons, and its motor domain represses microtubule polymerization in vitro. The crystal structure of the KLP-12 motor domain complexed with tubulin, which represents the high-resolution structural snapshot of the inhibition state of microtubule-end dynamics, revealed the bending effect of KLP-12 for tubulin. Comparison with the KIF5B-tubulin and KIF2C-tubulin complexes, which represent the elongation and shrinking forms of microtubule ends, respectively, showed the curvature of tubulin introduced by KLP-12 is in between them. Taken together, KLP-12 controls the proper length of axons by modulating the curvature of the microtubule ends to inhibit the microtubule dynamics.

From meter-long structures that allow nerve cells to stretch across a body to miniscule 'hairs' required for lung cells to clear mucus, many life processes rely on cells sporting projections which have the right size for their role. Networks of hollow filaments known as microtubules shape these structures and ensure that they have the appropriate dimensions. Controlling the length of microtubules is therefore essential for organisms, yet how this process takes place is still not fully elucidated. Previous research has shown that microtubules continue to grow when their end is straight but stop when it is curved. A family of molecular motors known as kinesin-4 participate in this process, but the exact mechanisms at play remain unclear. To investigate, Tuguchi, Nakano, Imasaki et al. focused on the KLP-12 protein, a kinesin-4 equivalent which helps to controls the length of microtubules in the tiny worm Caenorhabditis elegans. They performed genetic manipulations and imaged the interactions between KLP-12 and the growing end of a microtubule using X-ray crystallography. This revealed that KLP-12 controls the length of neurons by inhibiting microtubule growth. It does so by modulating the curvature of the growing end of the filament to suppress its extension. A 'snapshot' of KLP-12 binding to a microtubule at the resolution of the atom revealed exactly how the protein helps to bend the end of the filament to prevent it from growing further. These results will help to understand how nerve cells are shaped. This may also provide insights into the molecular mechanisms for various neurodegenerative disorders caused by problems with the human equivalents of KLP-12, potentially leading to new therapies.

Recruitment of Irgb6 to the membrane is a direct trigger for membrane deformation.

Irgb6 is a member of interferon γ-induced immunity related GTPase (IRG), and one of twenty "effector" IRGs, which coordinately attack parasitophorous vacuole membrane (PVM), causing death of intracellular pathogen. Although Irgb6 plays a pivotal role as a pioneer in the process of PVM disruption, the direct effect of Irgb6 on membrane remained to be elucidated. Here, we utilized artificial lipid membranes to reconstitute Irgb6-membrane interaction in vitro, and revealed that Irgb6 directly deformed the membranes. Liposomes incubated with recombinant Irgb6 were drastically deformed generating massive tubular protrusions in the absence of guanine nucleotide, or with GMP-PNP. Liposome deformation was abolished by incubating with Irgb6-K275A/R371A, point mutations at membrane targeting residues. The membrane tubules generated by Irgb6 were mostly disappeared by the addition of GTP or GDP, which are caused by detachment of Irgb6 from membrane. Binding of Irgb6 to the membrane, which was reconstituted in vitro using lipid monolayer, was stimulated at GTP-bound state. Irgb6 GTPase activity was stimulated by the presence of liposomes more than eightfold. Irgb6 GTPase activity in the absence of membrane was also slightly stimulated, by lowering ionic strength, or by increasing protein concentration, indicating synergistic stimulation of the GTPase activity. These results suggest that membrane targeting of Irgb6 and resulting membrane deformation does not require GTP, but converting into GTP-bound state is crucial for detaching Irgb6 from the membrane, which might coincident with local membrane disruption.

CAMSAP2 organizes a γ-tubulin-independent microtubule nucleation centre through phase separation.

Microtubules are dynamic polymers consisting of αß-tubulin heterodimers. The initial polymerization process, called microtubule nucleation, occurs spontaneously via αß-tubulin. Since a large energy barrier prevents microtubule nucleation in cells, the γ-tubulin ring complex is recruited to the centrosome to overcome the nucleation barrier. However, a considerable number of microtubules can polymerize independently of the centrosome in various cell types. Here, we present evidence that the minus-end-binding calmodulin-regulated spectrin-associated protein 2 (CAMSAP2) serves as a strong nucleator for microtubule formation by significantly reducing the nucleation barrier. CAMSAP2 co-condensates with αß-tubulin via a phase separation process, producing plenty of nucleation intermediates. Microtubules then radiate from the co-condensates, resulting in aster-like structure formation. CAMSAP2 localizes at the co-condensates and decorates the radiating microtubule lattices to some extent. Taken together, these in vitro findings suggest that CAMSAP2 supports microtubule nucleation and growth by organizing a nucleation centre as well as by stabilizing microtubule intermediates and growing microtubules.

Cells are able to hold their shape thanks to tube-like structures called microtubules that are made of hundreds of tubulin proteins. Microtubules are responsible for maintaining the uneven distribution of molecules throughout the cell, a phenomenon known as polarity that allows cells to differentiate into different types with various roles. A protein complex called the γ-tubulin ring complex (γ-TuRC) is necessary for microtubules to form. This protein helps bind the tubulin proteins together and stabilises microtubules. However, recent research has found that in highly polarized cells such as neurons, which have highly specialised regions, microtubules can form without γ-TuRC. Searching for the proteins that could be filling in for γ-TuRC in these cells some evidence has suggested that a group known as CAMSAPs may be involved, but it is not known how. To characterize the role of CAMSAPs, Imasaki, Kikkawa et al. studied how one of these proteins, CAMSAP2, interacts with tubulins. To do this, they reconstituted both CAMSAP2 and tubulins using recombinant biotechnology and mixed them in solution. These experiments showed that CAMSAP2 can help form microtubules by bringing together their constituent proteins so that they can bind to each other more easily. Once microtubules start to form, CAMSAP2 continues to bind to them, stabilizing them and enabling them to grow to full size. These results shed light on how polarity is established in cells such as neurons, muscle cells, and epithelial cells. Additionally, the ability to observe intermediate structures during microtubule formation can provide insights into the processes that these structures are involved in.

Structural basis of membrane recognition of Toxoplasma gondii vacuole by Irgb6.

The p47 immunity-related GTPase (IRG) Irgb6 plays a pioneering role in host defense against Toxoplasma gondii infection. Irgb6 is recruited to the parasitophorous vacuole membrane (PVM) formed by T. gondii and disrupts it. Despite the importance of this process, the molecular mechanisms accounting for PVM recognition by Irgb6 remain elusive because of lack of structural information on Irgb6. Here we report the crystal structures of mouse Irgb6 in the GTP-bound and nucleotide-free forms. Irgb6 exhibits a similar overall architecture to other IRGs in which GTP binding induces conformational changes in both the dimerization interface and the membrane-binding interface. The membrane-binding interface of Irgb6 assumes a unique conformation, composed of N- and C-terminal helical regions forming a phospholipid binding site. In silico docking of phospholipids further revealed membrane-binding residues that were validated through mutagenesis and cell-based assays. Collectively, these data demonstrate a novel structural basis for Irgb6 to recognize T. gondii PVM in a manner distinct from other IRGs.

Kinesin-binding-triggered conformation switching of microtubules contributes to polarized transport.

Kinesin-1, the founding member of the kinesin superfamily of proteins, is known to use only a subset of microtubules for transport in living cells. This biased use of microtubules is proposed as the guidance cue for polarized transport in neurons, but the underlying mechanisms are still poorly understood. Here, we report that kinesin-1 binding changes the microtubule lattice and promotes further kinesin-1 binding. This high-affinity state requires the binding of kinesin-1 in the nucleotide-free state. Microtubules return to the initial low-affinity state by washing out the binding kinesin-1 or by the binding of non-hydrolyzable ATP analogue AMPPNP to kinesin-1. X-ray fiber diffraction, fluorescence speckle microscopy, and second-harmonic generation microscopy, as well as cryo-EM, collectively demonstrated that the binding of nucleotide-free kinesin-1 to GDP microtubules changes the conformation of the GDP microtubule to a conformation resembling the GTP microtubule.

Structural insight into microtubule stabilization and kinesin inhibition by Tau family MAPs.

The Tau family microtubule-associated proteins (MAPs) promote microtubule stabilization and regulate microtubule-based motility. They share the C-terminal microtubule-binding domain, which includes three to five tubulin-binding repeats. Different numbers of repeats formed by alternative splicing have distinct effects on the activities of these proteins, and the distribution of these variants regulates fundamental physiological phenomena in cells. In this study, using cryo-EM, we visualized the MAP4 microtubule complex with the molecular motor kinesin-1. MAP4 bound to the C-terminal domains of tubulins along the protofilaments stabilizes the longitudinal contacts of the microtubule. The strongest bond of MAP4 was found around the intertubulin-dimer interface such that MAP4 coexists on the microtubule with kinesin-1 bound to the intratubulin-dimer interface as well. MAP4, consisting of five repeats, further folds and accumulates above the intertubulin-dimer interface, interfering with kinesin-1 movement. Therefore, these cryo-EM studies reveal new insight into the structural basis of microtubule stabilization and inhibition of kinesin motility by the Tau family MAPs.

Recent progress in structural biology: lessons from our research history.

The recent 'resolution revolution' in structural analyses of cryo-electron microscopy (cryo-EM) has drastically changed the research strategy for structural biology. In addition to X-ray crystallography and nuclear magnetic resonance spectroscopy, cryo-EM has achieved the structural analysis of biological molecules at near-atomic resolution, resulting in the Nobel Prize in Chemistry 2017. The effect of this revolution has spread within the biology and medical science fields affecting everything from basic research to pharmaceutical development by visualizing atomic structure. As we have used cryo-EM as well as X-ray crystallography since 2000 to elucidate the molecular mechanisms of the fundamental phenomena in the cell, here we review our research history and summarize our findings. In the first half of the review, we describe the structural mechanisms of microtubule-based motility of molecular motor kinesin by using a joint cryo-EM and X-ray crystallography method. In the latter half, we summarize our structural studies on transcriptional regulation by X-ray crystallography of in vitro reconstitution of a multi-protein complex.

Structural Insights into the Altering Function of CRMP2 by Phosphorylation.

Collapsin response mediator protein 2 (CRMP2) regulates neuronal polarity by controlling microtubule dynamics. CRMP2 activity is regulated by semaphorin-induced phosphorylation at the C-terminal tail domain. Unphosphorylated CRMP2 induces effective axonal microtubule formation to give the axonal characteristics to a neurite, whereas phosphorylated CRMP2 leads to the apparently opposite effect, growth cone collapse. We have recently characterized the structural detail of CRMP2-induced axonal microtubule formation (Niwa et al. (2017) Sci. Rep., 7: 10681). CRMP2 forms the hetero-trimer with GTP-tubulin to induce effective axonal microtubule formation in the future axon. Phosphorylation of CRMP2 has been reported to decrease the affinity between CRMP2 and the microtubule, albeit the molecular mechanisms of how the phosphorylation of CRMP2 changes the structure to achieve distinct effects from unphosphorylated CRMP2 is not well understood. Here we performed a series of biochemical and structural analyses of phospho-mimic CRMP2. Phosphorylation of CRMP2 undergoes small conformational changes at the C-terminal tail with shifting the surface charge, which not only alters the interactions within the CRMP2 tetramer but also alters the interactions with GTP-tubulin. Consequently, phospho-mimic CRMP2 fails to form a hetero-trimer with GTP-tubulin, thus losing the ability to establish and maintain the axonal microtubules.Key words: CRMP2, phosphorylation, microtubule, axon, crystal structure.

Structural basis for CRMP2-induced axonal microtubule formation.

Microtubule associated protein Collapsin response mediator protein 2 (CRMP2) regulates neuronal polarity in developing neurons through interactions with tubulins or microtubules. However, how CRMP2 promotes axonal formation by affecting microtubule behavior remains unknown. This study aimed to obtain the structural basis for CRMP2-tubulin/microtubule interaction in the course of axonogenesis. The X-ray structural studies indicated that the main interface to the soluble tubulin-dimer is the last helix H19 of CRMP2 that is distinct from the known C-terminal tail-mediated interaction with assembled microtubules. In vitro structural and functional studies also suggested that the H19-mediated interaction promoted the rapid formation of GTP-state microtubules directly, which is an important feature of the axon. Consistently, the H19 mutants disturbed axon elongation in chick neurons, and failed to authorize the structural features for axonal microtubules in Caenorhabditis elegans. Thus, CRMP2 induces effective axonal microtubule formation through H19-mediated interactions with a soluble tubulin-dimer allowing axonogenesis to proceed.

Motility and microtubule depolymerization mechanisms of the Kinesin-8 motor, KIF19A.

The kinesin-8 motor, KIF19A, accumulates at cilia tips and controls cilium length. Defective KIF19A leads to hydrocephalus and female infertility because of abnormally elongated cilia. Uniquely among kinesins, KIF19A possesses the dual functions of motility along ciliary microtubules and depolymerization of microtubules. To elucidate the molecular mechanisms of these functions we solved the crystal structure of its motor domain and determined its cryo-electron microscopy structure complexed with a microtubule. The features of KIF19A that enable its dual function are clustered on its microtubule-binding side. Unexpectedly, a destabilized switch II coordinates with a destabilized L8 to enable KIF19A to adjust to both straight and curved microtubule protofilaments. The basic clusters of L2 and L12 tether the microtubule. The long L2 with a characteristic acidic-hydrophobic-basic sequence effectively stabilizes the curved conformation of microtubule ends. Hence, KIF19A utilizes multiple strategies to accomplish the dual functions of motility and microtubule depolymerization by ATP hydrolysis.

Structural Basis of Backwards Motion in Kinesin-1-Kinesin-14 Chimera: Implication for Kinesin-14 Motility.

Kinesin-14 is a unique minus-end-directed microtubule-based motor. A swinging motion of a class-specific N-terminal neck helix has been proposed to produce minus-end directionality. However, it is unclear how swinging of the neck helix is driven by ATP hydrolysis utilizing the highly conserved catalytic core among all kinesins. Here, using a motility assay, we show that in addition to the neck helix, the conserved five residues at the C-terminal region in kinesin-14, namely the neck mimic, are necessary to give kinesin-1 an ability to reverse its directionality toward the minus end of microtubules. Our structural analyses further demonstrate that the C-terminal neck mimic, in cooperation with conformational changes in the catalytic core during ATP binding, forms a kinesin-14 bundle with the N-terminal neck helix to swing toward the minus end of microtubules. Thus, the neck mimic plays a crucial role in coupling the chemical ATPase reaction with the mechanical cycle to produce the minus-end-directed motility of kinesin-14.

X-ray and Cryo-EM structures reveal mutual conformational changes of Kinesin and GTP-state microtubules upon binding.

The molecular motor kinesin moves along microtubules using energy from ATP hydrolysis in an initial step coupled with ADP release. In neurons, kinesin-1/KIF5C preferentially binds to the GTP-state microtubules over GDP-state microtubules to selectively enter an axon among many processes; however, because the atomic structure of nucleotide-free KIF5C is unavailable, its molecular mechanism remains unresolved. Here, the crystal structure of nucleotide-free KIF5C and the cryo-electron microscopic structure of nucleotide-free KIF5C complexed with the GTP-state microtubule are presented. The structures illustrate mutual conformational changes induced by interaction between the GTP-state microtubule and KIF5C. KIF5C acquires the 'rigor conformation', where mobile switches I and II are stabilized through L11 and the initial portion of the neck-linker, facilitating effective ADP release and the weak-to-strong transition of KIF5C microtubule affinity. Conformational changes to tubulin strengthen the longitudinal contacts of the GTP-state microtubule in a similar manner to GDP-taxol microtubules. These results and functional analyses provide the molecular mechanism of the preferential binding of KIF5C to GTP-state microtubules.

New simulated annealing approach considering helix bending applied to determine the 8.8Å structure of 15-protofilament microtubules.

The helix is an important motif in biological architectures. The helical structures of nanoscale proteins are principally determined by three-dimensional (3D) reconstruction from electron micrographs. However, bending or distortion of flexible helices and the low contrast of the images recorded by cryo-electron microscopy, prevent the analysis from reaching high resolution. We have developed a novel helical reconstruction method that overcomes these issues, and present the processing of microtubule images to demonstrate its application. Cropping long helical structures into small square pieces allows bending or distortion of the helices to be accounted for. The initial image-frames are automatically positioned assuming perfect helical symmetry. A simulated annealing (SA)-based algorithm is then used to adjust the framing. This is guided by the contrast of 2D averages, which serve as an accuracy index. After the initial 3D reconstruction, the position and orientation of each average image is iteratively adjusted to give the best match between the input average and the reprojection from the reconstruction. Finally, reconstructions from images recorded at different defocus values, are aligned and averaged to compensate the contrast transfer modulation and improve the resolution. The method successfully determined the structure of a 15-protofilament microtubule. The 8.8Å resolution (7.8Å using the 0.143 FSC criterion) attained allows differences between the α- and ß- tubulins to be discerned in the absence of a molecular landmark such as microtubule-associated proteins, for the first time by electron microscopy. The SA-based method is applicable to other helical protein complexes and in general to helical structures.

Structural basis for the ATP-induced isomerization of kinesin.

Kinesin superfamily proteins (KIFs) are microtubule-based molecular motors driven by the energy derived from the hydrolysis of ATP. Previous studies have revealed that the ATP binding step is crucial both for the power stroke to produce motility and for the inter-domain regulation of ATPase activity to guarantee the processive movement of dimeric KIFs. Here, we report the first crystal structure of KIF4 complexed with the non-hydrolyzable ATP analog, AMPPNP (adenylyl imidodiphosphate), at 1.7Å resolution. By combining our structure with previously solved KIF1A structures complexed with two ATP analogs, molecular snapshots during ATP binding reveal that the closure of the nucleotide-binding pocket during ATP binding is achieved by closure of the backdoor. Closure of the backdoor stabilizes two mobile regions, switch I and switch II, to generate the phosphate tube from which hydrolyzed phosphate is released. Through the stabilization of switch II, the local conformational change at the catalytic center is further relayed to the neck-linker element that fully docks to the catalytic core to produce the power stroke. Because the neck linker is a sole element that connects the partner heads in dimeric KIFs, this tight structural coordination between the catalytic center and neck linker enables inter-domain communication between the partner heads. This study also revealed the putative microtubule-binding site of KIF4, thus providing structural insights that describe the specific binding of KIF4 to the microtubule.

Conformational changes in tubulin in GMPCPP and GDP-taxol microtubules observed by cryoelectron microscopy.

Microtubules are dynamic polymers that stochastically switch between growing and shrinking phases. Microtubule dynamics are regulated by guanosine triphosphate (GTP) hydrolysis by ß-tubulin, but the mechanism of this regulation remains elusive because high-resolution microtubule structures have only been revealed for the guanosine diphosphate (GDP) state. In this paper, we solved the cryoelectron microscopy (cryo-EM) structure of microtubule stabilized with a GTP analogue, guanylyl 5'-α,ß-methylenediphosphonate (GMPCPP), at 8.8-Å resolution by developing a novel cryo-EM image reconstruction algorithm. In contrast to the crystal structures of GTP-bound tubulin relatives such as γ-tubulin and bacterial tubulins, significant changes were detected between GMPCPP and GDP-taxol microtubules at the contacts between tubulins both along the protofilament and between neighboring protofilaments, contributing to the stability of the microtubule. These findings are consistent with the structural plasticity or lattice model and suggest the structural basis not only for the regulatory mechanism of microtubule dynamics but also for the recognition of the nucleotide state of the microtubule by several microtubule-binding proteins, such as EB1 or kinesin.

The mechanisms of kinesin motor motility: lessons from the monomeric motor KIF1A.

Most kinesins move processively along microtubules by using energy derived from ATP hydrolysis. Almost all of the intermediate structures of this ATPase reaction cycle have been solved for the monomeric kinesin 3 family motor KIF1A. Based on this structural information, we propose a common mechanism of kinesin motility, focusing on the regulation of kinesin motility through their interaction with microtubules and by their 'neck-linker' region, which connects their motor domain to cargo and kinesin partner heads.