The kinesin-9 family comprises two subfamilies specific to ciliated eukaryotic cells, and has recently attracted considerable attention because of its importance in ciliary bending and formation. However, only scattered data are available on the motor properties of kinesin-9 family members; these properties have not been compared under identical experimental conditions using kinesin-9 motors from the same species. Here, we report the comprehensive motor properties of two kinesin-9 molecules of Tetrahymena thermophila, TtK9A (Kif9/Klp1 ortholog) and TtK9B1 (Kif6 ortholog), using microtubule-based in vitro assays, including single-motor and multi-motor assays and microtubule-stimulated ATPase assays. Both subfamilies exhibit microtubule plus-end-directed, extremely slow motor activity, both in single and multiple molecules. TtK9A shows lower processivity than TtK9B1. Our findings indicate that the considerable slow movement of kinesin-9 that corresponds to low ATP hydrolysis rates is a common feature of the ciliary kinesin-9 family.

Plant myosins have higher velocities than animal myosins. Among them, myosins in freshwater algae of the genus Chara have extremely high velocities. We have biochemically studied myosins that perform high-speed movements in the alga Chara. Our studies have elucidated the structural and enzymatic basis for the fast movement of Chara myosins. This review outlines the history leading to the discovery of the fastest myosin, algae Chara myosin XI, and the structure-function correlation of the fastest myosin. This review article is an extended version of the Japanese article, \"Structure-function Relationship of the Fastest Myosin\" by Ito et al., published in SEIBUTSU BUTSURI Vol. 63, p. 91-96 (2023).

Axonemal dyneins are the driving force of motile cilia, while cytoplasmic dyneins play an essential role in minus-end oriented intracellular transport. Their molecular structure is indispensable for an understanding of the molecular mechanism of ciliary beating and cargo transport. After some initial structural analysis of cytoplasmic dyneins, which are easier to manipulate with genetic engineering, using X-ray crystallography and single-particle cryo-electron microscopy, a number of atomic and pseudo-atomic structural analyses of axonemal dyneins have been published. Currently, several structures of dyneins in the post-power stroke conformation as well as a few structures in the pre-power stroke conformation are available. It will be worth systematically comparing conformations of dynein motor proteins from different sources and at different states, to understand their role in biological function. In this review, we will overview published high- and intermediate-resolution structures of cytoplasmic and axonemal dyneins, compare the high-resolution structures of their core motor domains and overall tail conformations at various nucleotide states, and discuss their force generation mechanism.

Glial cells provide physical and chemical support and protection for neurons and for the extracellular compartments of neural tissue through secretion of soluble factors, insoluble scaffolds, and vesicles. Additionally, glial cells have regenerative capacity by remodeling their physical microenvironment and changing physiological properties of diverse cell types in their proximity. Various types of aberrant glial and macrophage cells are associated with human diseases, disorders, and malignancy. We previously demonstrated that transmembrane protein, TMEM230 has tissue revascularization and regenerating capacity by its ability to secrete pro-angiogenic factors and metalloproteinases, inducing endothelial cell sprouting and channel formation. In healthy normal neural tissue, TMEM230 is predominantly expressed in glial and marcophate cells, suggesting a prominent role in neural tissue homeostasis. TMEM230 regulation of the endomembrane system was supported by co-expression with RNASET2 (lysosome, mitochondria, and vesicles) and STEAP family members (Golgi complex). Intracellular trafficking and extracellular secretion of glial cellular components are associated with endocytosis, exocytosis and phagocytosis mediated by motor proteins. Trafficked components include metalloproteins, metalloproteinases, glycans, and glycoconjugate processing and digesting enzymes that function in phagosomes and vesicles to regulate normal neural tissue microenvironment, homeostasis, stress response, and repair following neural tissue injury or degeneration. Aberrantly high sustained levels TMEM230 promotes metalloprotein expression, trafficking and secretion which contribute to tumor associated infiltration and hypervascularization of high tumor grade gliomas. Following injury of the central nervous or peripheral systems, transcient regulated upregulation of TMEM230 promotes tissue wound healing, remodeling and revascularization by activating glial and macrophage generated microchannels/microtubules (referred to as vascular mimicry) and blood vessel sprouting and branching. Our results support that TMEM230 may act as a master regulator of motor protein mediated trafficking and compartmentalization of a large class of metalloproteins in gliomas and gliosis.

Kinesins are eukaryotic microtubule motor proteins subdivided into conserved families with distinct functional roles. While many kinesin families are widespread in eukaryotes, each organismal lineage maintains a unique kinesin repertoire composed of many families with distinct numbers of genes. Previous genomic surveys indicated that land plant kinesin repertoires differ markedly from other eukaryotes. To determine when repertoires diverged during plant evolution, we performed robust phylogenomic analyses of kinesins in 24 representative plants, two algae, two animals, and one yeast. These analyses show that kinesin repertoires expand and contract coincident with major shifts in the biology of algae and land plants. One kinesin family and five subfamilies, each defined by unique domain architectures, emerged in the green algae. Four of those kinesin groups expanded in ancestors of modern land plants, while six other kinesin groups were lost in the ancestors of pollen-bearing plants. Expansions of different kinesin families and subfamilies occurred in moss and angiosperm lineages. Other kinesin families remained stable and did not expand throughout plant evolution. Collectively these data support a radiation of kinesin domain architectures in algae followed by differential positive and negative selection on kinesins families and subfamilies in different lineages of land plants.

In eukaryotes, the ubiquitin-proteasome system is responsible for intracellular protein degradation. Proteins tagged with ubiquitin are recognized by ubiquitin receptors on the 19S regulatory particle (RP) of the 26S proteasome, unfolded, routed through the translocation channel of the RP, and are then degraded in the 20S core particle (CP). Aromatic paddles on the pore-1 loops of the RP\'s Rpt subunits grip the substrate and pull folded domains into the channel, thereby unfolding them. The sequence that the aromatic paddles grip while unfolding a substrate is therefore expected to influence the extent of unfolding, and low complexity sequences have been shown to interfere with grip. However, the detailed spatial requirements for grip while unfolding proteins, particularly from the N-terminus, remain unknown. We determined how the location of glycine-rich tracts relative to a folded domain impairs unfolding. We find that, in contrast to a previous report, inserting glycine-rich sequences closer to the folded domain reduced unfolding ability more than positioning them further away. Locations that have the biggest effect on unfolding map onto the regions where the aromatic paddles are predicted to interact with the substrate. Effects on unfolding from locations up to 67 amino acids away from the folded domain suggest that there are additional interactions between the substrate and the proteasome beyond the aromatic paddles that facilitate translocation of the substrate. In sum, this study deepens understanding of the mechanical interactions within the substrate channel by mapping the spacing of interactions between the substrate and the proteasome during unfolding.

Myosins are important motor proteins that associate with the actin cytoskeleton. Structurally, myosins function as heteromeric complexes where smaller light chains, such as calmodulin (CaM), bind to isoleucine-glutamine (IQ) domains in the neck region to facilitate mechano-enzymatic activity. We recently identified Arabidopsis CaM-like (CML) proteins CML13 and CML14 as interactors of proteins containing multiple IQ domains, including a myosin VIII. Here, we demonstrate that CaM, CML13, and CML14 bind the neck region of all four Arabidopsis myosin VIII isoforms. Among CMLs tested for binding to myosins VIIIs, CaM, CML13, and CML14 gave the strongest signals using in planta split-luciferase protein interaction assays. In vitro, recombinant CaM, CML13, and CML14 showed specific, high-affinity, calcium-independent binding to the IQ domains of myosin VIIIs. CaM, CML13, and CML14 co-localized to plasma membrane-bound puncta when co-expressed with red fluorescent protein-myosin fusion proteins containing IQ and tail domains of myosin VIIIs. In vitro actin motility assays using recombinant myosin VIIIs demonstrated that CaM, CML13, and CML14 function as light chains. Suppression of CML13 or CML14 expression using RNA silencing resulted in a shortened-hypocotyl phenotype, similar to that observed in a quadruple myosin mutant, myosin viii4KO. Collectively, our data indicate that Arabidopsis CML13 and CML14 are novel myosin VIII light chains.

In this study, we consider axonal transport of large cargo vesicles characterised by transient expansion of the axon shaft. Our goal is to formulate a mathematical model which captures the dynamic mechanical interaction of such cargo vesicles with the membrane associated periodic cytoskeletal structure (MPS). It consists of regularly spaced actin rings that are transversal to the longitudinal direction of the axon and involved in the radial contraction of the axon. A system of force balance equations is formulated by which we describe the transversal rings as visco-elastic Kelvin-Voigt elements. In a homogenisation limit, we reformulate the model as a free boundary problem for the interaction of the submembranous MPS with the large vesicle. We derive a non-linear force-velocity relation as a quasi-steady state solution. Computationally we analyse the vesicle size dependence of the transport speed and use an asymptotic approximation to formulate it as a power law that can be tested experimentally.

Conventional kinesin-1 is the primary anterograde motor in cells for transporting cellular cargo. While there is a consensus that the C-terminal tail of kinesin-1 inhibits motility, the molecular architecture of a full-length autoinhibited kinesin-1 remains unknown. Here, we combine crosslinking mass spectrometry (XL-MS), electron microscopy (EM), and AlphaFold structure prediction to determine the architecture of the full-length autoinhibited kinesin-1 homodimer (kinesin-1 heavy chain [KHC]) and kinesin-1 heterotetramer (KHC bound to kinesin light chain 1 [KLC1]). Our integrative analysis shows that kinesin-1 forms a compact, bent conformation through a break in coiled-coil 3. Moreover, our XL-MS analysis demonstrates that kinesin light chains stabilize the folded inhibited state rather than inducing a new structural state. Using our structural model, we show that disruption of multiple interactions between the motor, stalk, and tail domains is required to activate the full-length kinesin-1. Our work offers a conceptual framework for understanding how cargo adaptors and microtubule-associated proteins relieve autoinhibition to promote activation.

Rods and cones are photoreceptor neurons in the retina that are required for visual sensation in vertebrates, where proper protein localization and compartmentalization are critical for phototransduction and visual function. In human retinal diseases, improper protein transport to the outer segment (OS) or mislocalization of proteins to the inner segment (IS) could lead to impaired visual responses and photoreceptor cell degeneration, causing a loss of visual function. We showed involvement of an unconventional motor protein, MYO1C, in the proper localization of rhodopsin to the OS, where loss of MYO1C in a mammalian model caused mislocalization of rhodopsin to IS and cell bodies, leading to progressively severe retinal phenotypes. In this study, using modeling and docking analysis, we aimed to identify the protein-protein interaction sites between MYO1C and Rhodopsin to establish a hypothesis that a physical interaction between these proteins is necessary for the proper trafficking of rhodopsin and visual function.