GSK3β phosphorylation modulates CLASP–microtubule association and lamella microtubule attachment

Polarity of the microtubule (MT) cytoskeleton is essential for many cell functions. Cytoplasmic linker–associated proteins (CLASPs) are MT-associated proteins thought to organize intracellular MTs and display a unique spatiotemporal regulation. In migrating epithelial cells, CLASPs track MT plus ends in the cell body but bind along MTs in the lamella. In this study, we demonstrate that glycogen synthase kinase 3β (GSK3β) directly phosphorylates CLASPs at multiple sites in the domain required for MT plus end tracking. Although complete phosphorylation disrupts both plus end tracking and association along lamella MTs, we show that partial phosphorylation of the identified GSK3β motifs determines whether CLASPs track plus ends or associate along MTs. In addition, we find that expression of constitutively active GSK3β destabilizes lamella MTs by disrupting lateral MT interactions with the cell cortex. GSK3β-induced lamella MT destabilization was partially rescued by expression of CLASP2 with mutated phosphorylation sites. This indicates that CLASP-mediated stabilization of peripheral MTs, which likely occurs in the vicinity of focal adhesions, may be regulated by local GSK3β inactivation.     

Microtubule nucleation at the cis‐side of the Golgi apparatus requires AKAP450 and GM130

We report that microtubule (MT) nucleation at the Golgi apparatus requires AKAP450, a centrosomal γ‐TuRC‐interacting protein that also forms a distinct network associated with the Golgi. Depletion of AKAP450 abolished MT nucleation at the Golgi, whereas depletion of the cis‐Golgi protein GM130 led to the disorganisation of AKAP450 network and impairment of MT nucleation. Brefeldin‐A treatment induced relocalisation of AKAP450 to ER exit sites and concomitant redistribution of MT nucleation capacity to the ER. AKAP450 specifically binds the cis‐side of the Golgi in an MT‐independent, GM130‐dependent manner. Short AKAP450‐dependent growing MTs are covered by CLASP2. Like for centrosome, dynein/dynactin complexes are necessary to anchor MTs growing from the Golgi. We further show that Golgi‐associated AKAP450 has a role in cell migration rather than in cell polarisation of the centrosome–Golgi apparatus. We propose that the recruitment of AKAP450 on the Golgi membranes through GM130 allows centrosome‐associated nucleating activity to extend to the Golgi, to control the assembly of subsets of MTs ensuring specific functions within the Golgi or for transporting specific cargos to the cell periphery.     

The centrosome–Golgi apparatus nexus

A shared feature among all microtubule (MT)-dependent processes is the requirement for MTs to be organized in arrays of defined geometry. At a fundamental level, this is achieved by precisely controlling the timing and localization of the nucleation events that give rise to new MTs. To this end, MT nucleation is restricted to specific subcellular sites called MT-organizing centres. The primary MT-organizing centre in proliferating animal cells is the centrosome. However, the discovery of MT nucleation capacity of the Golgi apparatus (GA) has substantially changed our understanding of MT network organization in interphase cells. Interestingly, MT nucleation at the Golgi apparently relies on multiprotein complexes, similar to those present at the centrosome, that assemble at the cis-face of the organelle. In this process, AKAP450 plays a central role, acting as a scaffold to recruit other centrosomal proteins important for MT generation. MT arrays derived from either the centrosome or the GA differ in their geometry, probably reflecting their different, yet complementary, functions. Here, I review our current understanding of the molecular mechanisms involved in MT nucleation at the GA and how Golgi- and centrosome-based MT arrays work in concert to ensure the formation of a pericentrosomal polarized continuous Golgi ribbon structure, a critical feature for cell polarity in mammalian cells. In addition, I comment on the important role of the Golgi-nucleated MTs in organizing specialized MT arrays that serve specific functions in terminally differentiated cells.     

γ-Tubulin ring complexes regulate microtubule plus end dynamics

γ-Tubulin is critical for the initiation and regulation of microtubule (MT) assembly. In Drosophila melanogaster, it acts within two main complexes: the γ-tubulin small complex (γ-TuSC) and the γ-tubulin ring complex (γ-TuRC). Proteins specific of the γ-TuRC, although nonessential for viability, are required for efficient mitotic progression. Until now, their role during interphase remained poorly understood. Using RNA interference in Drosophila S2 cells, we show that the γ-TuRC is not critical for overall MT organization. However, depletion of any component of this complex results in an increase of MT dynamics. Combined immunofluorescence and live imaging analysis allows us to reveal that the γ-TuRC localizes along interphase MTs and that distal γ-tubulin spots match with sites of pause or rescue events. We propose that, in addition to its role in nucleation, the γ-TuRC associated to MTs may regulate their dynamics by limiting catastrophes.     

The γ -Tubulin Complex Protein GCP4 Is Required for Organizing Functional Microtubule Arrays in Arabidopsis thaliana

Microtubule (MT) nucleation and organization depend on the evolutionarily conserved protein γ -tubulin, which forms a complex with GCP2-GCP6 (GCP for γ -Tubulin Complex Protein). To date, it is still unclear how GCP4-GCP6 (the non-core GCPs) may be involved in acentrosomal MT nucleation in plant cells. We found that GCP4 was associated with γ -tubulin in vivo in Arabidopsis thaliana. When GCP4 expression was repressed by an artificial microRNA, transgenic plants exhibited phenotypes of dwarfism and reduced organ size. In mitotic cells, it was observed that the γ -tubulin signal associated with the mitotic spindle, and the phragmoplast was depleted when GCP4 was downregulated. Consequently, MTs failed to converge at unified spindle poles, and the bipolar phragmoplast MT array frequently had discrete bundles with extended minus ends, resulting in failed cytokinesis as reflected by cell wall stubs in leaf epidermal cells. In addition, cortical MTs in swollen guard cells and pavement cells of the leaf epidermis became hyperparallel and bundled, which was likely caused by frequent MT nucleation with shallow angles on the wall of extant MTs. Therefore, our results support the notion that GCP4 is an indispensable component for the function of γ -tubulin in MT nucleation and organization in plant cells.     

Dystrophin is a microtubule-associated protein

Cytolinkers are giant proteins that can stabilize cells by linking actin filaments, intermediate filaments, and microtubules (MTs) to transmembrane complexes. Dystrophin is functionally similar to cytolinkers, as it links the multiple components of the cellular cytoskeleton to the transmembrane dystroglycan complex. Although no direct link between dystrophin and MTs has been documented, costamere-associated MTs are disrupted when dystrophin is absent. Using tissue-based cosedimentation assays on mice expressing endogenous dystrophin or truncated transgene products, we find that constructs harboring spectrinlike repeat 24 through the first third of the WW domain cosediment with MTs. Purified Dp260, a truncated isoform of dystrophin, bound MTs with a K_d of 0.66 µM, a stoichiometry of 1 Dp260/1.4 tubulin heterodimer at saturation, and stabilizes MTs from cold-induced depolymerization. Finally, α- and β-tubulin expression is increased ∼2.5-fold in mdx skeletal muscle without altering the tubulin–MT equilibrium. Collectively, these data suggest dystrophin directly organizes and/or stabilizes costameric MTs and classifies dystrophin as a cytolinker in skeletal muscle.     

In Vitro Reconstitution of Microtubule Plus End-directed, GTPγS-sensitive Motility of Golgi Membranes

Purified Golgi membranes were mixed with cytosol and microtubules (MTs) and observed by video enhanced light microscopy. Initially, the membranes appeared as vesicles that moved along MTs. As time progressed, vesicles formed aggregates from which membrane tubules emerged, traveled along MTs, and eventually generated extensive reticular networks. Membrane motility required ATP, occurred mainly toward MT plus ends, and was inhibited almost completely by the H1 monoclonal antibody to kinesin heavy chain, 5′-adenylylimidodiphosphate, and 100 μM but not 20 μM vanadate. Motility was also blocked by GTPγS or AlF₄⁻ but was insensitive to AlCl₃, NaF, staurosporin, or okadaic acid. The targets for GTPγS and AlF₄⁻ were evidently of cytosolic origin, did not include kinesin or MTs, and were insensitive to several probes for trimeric G proteins. Transport of Golgi membranes along MTs mediated by a kinesin has thus been reconstituted in vitro. The motility is regulated by one or more cytosolic GTPases but not by protein kinases or phosphatases that are inhibited by staurosporin or okadaic acid, respectively. The pertinent GTPases are likely to be small G proteins or possibly dynamin. The in vitro motility may correspond to Golgi-to-ER or Golgi-to-cell surface transport in vivo.     

Modularity in Protein Evolution: Modular Organization and De Novo Domain Evolution in Mollusk Metallothioneins

Metallothioneins (MTs) are proteins devoted to the control of metal homeostasis and detoxification, and therefore, MTs have been crucial for the adaptation of the living beings to variable situations of metal bioavailability. The evolution of MTs is, however, not yet fully understood, and to provide new insights into it, we have investigated the MTs in the diverse classes of Mollusks. We have shown that most molluskan MTs are bimodular proteins that combine six domains—α, β1, β2, β3, γ, and δ—in a lineage-specific manner. We have functionally characterized the Neritimorpha β₃β₁ and the Patellogastropoda γβ₁ MTs, demonstrating the metal-binding capacity of the new γ domain. Our results have revealed a modular organization of mollusk MT, whose evolution has been impacted by duplication, loss, and de novo emergence of domains. MTs represent a paradigmatic example of modular evolution probably driven by the structural and functional requirements of metal binding.     

TIP150 interacts with and targets MCAK at the microtubule plus ends

The microtubule (MT) cytoskeleton orchestrates the cellular plasticity and dynamics that underlie morphogenesis and cell division. Growing MT plus ends have emerged as dynamic regulatory machineries in which specialized proteins—called plus-end tracking proteins (+TIPs)—bind to and control the plus-end dynamics that are essential for cell division and migration. However, the molecular mechanisms underlying the plus-end regulation by +TIPs at spindle and astral MTs have remained elusive. Here, we show that TIP150 is a new +TIP that binds to end-binding protein 1 (EB1) in vitro and co-localizes with EB1 at the MT plus ends in vivo. Suppression of EB1 eliminates the plus-end localization of TIP150. Interestingly, TIP150 also binds to mitotic centromere-associated kinesin (MCAK), an MT depolymerase that localizes to the plus end of MTs. Suppression of TIP150 diminishes the plus-end localization of MCAK. Importantly, aurora B-mediated phosphorylation disrupts the TIP150–MCAK association in vitro. We reason that TIP150 facilitates the EB1-dependent loading of MCAK onto MT plus ends and orchestrates the dynamics at the plus end of MTs.     

Microtubule Severing by Katanin p60 AAA+ ATPase Requires the C-terminal Acidic Tails of Both α- and β-Tubulins and Basic Amino Acid Residues in the AAA+ Ring Pore

The microtubule (MT) network is highly dynamic and undergoes dramatic reorganizations during the cell cycle. Dimers of α- and β-tubulins rapidly polymerize to and depolymerize from the end of MT fibrils in an intrinsic GTP-dependent manner. MT severing by ATP-driven enzymes such as katanin and spastin contributes significantly to microtubule dynamics, and it has been shown that katanin p60, a AAA+ family protein, has ATPase and MT-severing activities. The mechanism of MT severing by katanin p60 is poorly understood, and the residues in katanin p60 and tubulins important for severing activity were therefore explored in this study. MT-severing activity, but not ATPase activity, was inhibited by mutations of the conserved aromatic residue and the flanking basic residues in the pore region of the katanin p60 hexameric ring. When the acidic residue-rich C-terminal unstructured segment of either α- or β-tubulin was removed, polymerized MTs were resistant to katanin p60 treatment. Interactions between katanin p60 and the mutant MTs, on the other hand, were unaffected. Taken together, these findings led us to propose that the interactions between the positively charged residues of katanin p60 and the acidic tails of both tubulins are essential for efficient severing of MTs.     

Microtubule-associated proteins promote microtubule generation in the absence of γ-tubulin in human colon cancer cells

The γ-tubulin complex acts as the predominant microtubule (MT) nucleator that initiates MT formation and is therefore an essential factor for cell proliferation. Nonetheless, cellular MTs are formed after experimental depletion of the γ-tubulin complex, suggesting that cells possess other factors that drive MT nucleation. Here, by combining gene knockout, auxin-inducible degron, RNA interference, MT depolymerization/regrowth assay, and live microscopy, we identified four microtubule-associated proteins (MAPs), ch-TOG, CLASP1, CAMSAPs, and TPX2, which are involved in γ-tubulin–independent MT generation in human colon cancer cells. In the mitotic MT regrowth assay, nucleated MTs organized noncentriolar MT organizing centers (ncMTOCs) in the absence of γ-tubulin. Depletion of CLASP1 or TPX2 substantially delayed ncMTOC formation, suggesting that these proteins might promote MT nucleation in the absence of γ-tubulin. In contrast, depletion of ch-TOG or CAMSAPs did not affect the timing of ncMTOC appearance. CLASP1 also accelerates γ-tubulin–independent MT regrowth during interphase. Thus, MT generation can be promoted by MAPs without the γ-tubulin template.     

FAM29A promotes microtubule amplification via recruitment of the NEDD1–γ-tubulin complex to the mitotic spindle

Microtubules (MTs) are nucleated from centrosomes and chromatin. In addition, MTs can be generated from preexiting MTs in a γ-tubulin–dependent manner in yeast, plant, and Drosophila cells, although the underlying mechanism remains unknown. Here we show the spindle-associated protein FAM29A promotes MT-dependent MT amplification and is required for efficient chromosome congression and segregation in mammalian cells. Depletion of FAM29A reduces spindle MT density. FAM29A is not involved in the nucleation of MTs from centrosomes and chromatin, but is required for a subsequent increase in MT mass in cells released from nocodazole. FAM29A interacts with the NEDD1–γ-tubulin complex and recruits this complex to the spindle, which, in turn, promotes MT polymerization. FAM29A preferentially associates with kinetochore MTs and knockdown of FAM29A reduces the number of MTs in a kinetochore fiber, activates the spindle checkpoint, and delays the mitotic progression. Our study provides a biochemical mechanism for MT-dependent MT amplification and for the maturation of kinetochore fibers in mammalian cells.     

αTubulin 67C and Ncd Are Essential for Establishing a Cortical Microtubular Network and Formation of the Bicoid mRNA Gradient in Drosophila

The Bicoid (Bcd) protein gradient in Drosophila serves as a paradigm for gradient formation in textbooks. To explain the generation of the gradient, the ARTS model, which is based on the observation of a bcd mRNA gradient, proposes that the bcd mRNA, localized at the anterior pole at fertilization, migrates along microtubules (MTs) at the cortex to the posterior to form a bcd mRNA gradient which is translated to form a protein gradient. To fulfil the criteria of the ARTS model, an early cortical MT network is thus a prerequisite. We report hitherto undiscovered MT activities in the early embryo important for bcd mRNA transport: (i) an early and omnidirectional MT network exclusively at the anterior cortex of early nuclear cycle embryos showing activity during metaphase and anaphase only, (ii) long MTs up to 50 µm extending into the yolk at blastoderm stage to enable basal-apical transport. The cortical MT network is not anchored to the actin cytoskeleton. The posterior transport of the mRNA via the cortical MT network critically depends on maternally-expressed αTubulin67C and the minus-end motor Ncd. In either mutant, cortical transport of the bcd mRNA does not take place and the mRNA migrates along another yet undisclosed interior MT network, instead. Our data strongly corroborate the ARTS model and explain the occurrence of the bcd mRNA gradient.     

Molecular motors are differentially distributed on Golgi membranes from polarized epithelial cells

Microtubules (MT) are required for the efficient transport of membranes from the trans-Golgi and for transcytosis of vesicles from the basolateral membrane to the apical cytoplasm in polarized epithelia. MTs in these cells are primarily oriented with their plus ends basally near the Golgi and their minus-ends in the apical cytoplasm. Here we report that isolated Golgi and Golgi-enriched membranes from intestinal epithelial cells possess the actin based motor myosin-I, the MT minus- end-directed motor cytoplasmic dynein and its in vitro motility activator dynactin (p150/Glued). The Golgi can be separated into stacks, possessing features of the Golgi cisternae, and small membranes enriched in the trans-Golgi network marker TGN 38/41. Whereas myosin-I is present on all membranes in the Golgi fraction, dynein is present only on the small membrane fraction. Dynein, like myosin-I, is associated with membranes as a cytoplasmic peripheral membrane protein. Dynein and myosin-I coassociate with membranes that bind to MTs and cross-link actin filaments and MTs in a nucleotide-dependent manner. We propose that cytoplasmic dynein moves Golgi membranes along MTs to the cell cortex where myosin-I provides local delivery through the actin- rich cytoskeleton to the apical membrane.     

A Highlights from MBoC Selection: MAP65/Ase1 promote microtubule flexibility

Microtubules (MTs) are dynamic cytoskeletal elements involved in numerous cellular processes. Although they are highly rigid polymers with a persistence length of 1–8 mm, they may exhibit a curved shape at a scale of few micrometers within cells, depending on their biological functions. However, how MT flexural rigidity in cells is regulated remains poorly understood. Here we ask whether MT-associated proteins (MAPs) could locally control the mechanical properties of MTs. We show that two major cross-linkers of the conserved MAP65/PRC1/Ase1 family drastically decrease MT rigidity. Their MT-binding domain mediates this effect. Remarkably, the softening effect of MAP65 observed on single MTs is maintained when MTs are cross-linked. By reconstituting physical collisions between growing MTs/MT bundles, we further show that the decrease in MT stiffness induced by MAP65 proteins is responsible for the sharp bending deformations observed in cells when they coalign at a steep angle to create bundles. Taken together, these data provide new insights into how MAP65, by modifying MT mechanical properties, may regulate the formation of complex MT arrays.     

Disruption of the Golgi apparatus with brefeldin A does not destabilize the associated detyrosinated microtubule network.

Stable subsets of microtubules (MTs) are often enriched in detyrosinated alpha-tubulin. Recently it has been found that the Golgi apparatus is associated with a subset of relatively stable MTs and that detyrosinated MTs colocalize spatially and temporally with the Golgi apparatus in several cell lines. To determine whether the Golgi apparatus actively stabilizes associated MTs and thus allows their time-dependent detyrosination, we have used the drug brefeldin A (BFA) to disrupt the Golgi apparatus and have monitored changes in the Golgi apparatus and MT populations using simultaneous immunofluorescence and fluorescent lectin microscopy. We found that although BFA caused the Golgi apparatus to completely redistribute to the endoplasmic reticulum (ER), the detyrosinated MTs were not disrupted and remained in a juxtanuclear region. By Western blot analysis we found that even after 6 h of continuous exposure of cells to BFA, there was no detectable reduction in the level of detyrosinated alpha-tubulin. Simultaneous treatment with nocodazole and BFA led to a complete disruption of all MTs and normal Golgi structure/organization. Upon removal of nocodazole in the continued presence of BFA, we found that the detyrosinated MTs reformed in a compact juxtanuclear location in the absence of an intact Golgi complex. Finally, we found that the detyrosinated MTs colocalized precisely with a BFA-resistant structure that binds to the lectin, wheat germ agglutinin. We conclude that the juxtanuclear detyrosinated MTs are not actively stabilized by association with BFA-sensitive Golgi membranes. However, another closely associated structure which binds wheat germ agglutinin may serve to stabilize the juxtanuclear MTs. Alternatively, the MT organizing center (MTOC) and/or MT-associated proteins (MAPs) may organize and stabilize the juxtanuclear detyrosinated MTs.     

A Refined Reaction-Diffusion Model of Tau-Microtubule Dynamics and Its Application in FDAP Analysis

Fluorescence decay after photoactivation (FDAP) and fluorescence recovery after photobleaching (FRAP) are well established approaches for studying the interaction of the microtubule (MT)-associated protein tau with MTs in neuronal cells. Previous interpretations of FDAP/FRAP data have revealed dwell times of tau on MTs in the range of several seconds. However, this is difficult to reconcile with a dwell time recently measured by single-molecule analysis in neuronal processes that was shorter by two orders of magnitude. Questioning the validity of previously used phenomenological interpretations of FDAP/FRAP data, we have generalized the standard two-state reaction-diffusion equations by 1), accounting for the parallel and discrete arrangement of MTs in cell processes (i.e., homogeneous versus heterogeneous distribution of tau-binding sites); and 2), explicitly considering both active (diffusion upon MTs) and passive (piggybacking upon MTs at rates of slow axonal transport) motion of bound tau. For some idealized cases, analytical solutions were derived. By comparing them with the full numerical solution and Monte Carlo simulations, the respective validity domains were mapped. Interpretation of our FDAP data (from processes of neuronally differentiated PC12 cells) in light of the heterogeneous formalism yielded independent estimates for the association (∼2 ms) and dwell (∼100 ms) times of tau to/on a single MT rather than in an MT array. The dwell time was shorter by orders of magnitude than that in a previous report where a homogeneous topology of MTs was assumed. We found that the diffusion of bound tau was negligible in vivo, in contrast to an earlier report that tau diffuses along the MT lattice in vitro. Methodologically, our results demonstrate that the heterogeneity of binding sites cannot be ignored when dealing with reaction-diffusion of cytoskeleton-associated proteins. Physiologically, the results reveal the behavior of tau in cellular processes, which is noticeably different from that in vitro.     

Motor-dependent microtubule disassembly driven by tubulin tyrosination

In cells, stable microtubules (MTs) are covalently modified by a carboxypeptidase, which removes the C-terminal Tyr residue of α-tubulin. The significance of this selective detyrosination of MTs is not understood. In this study, we report that tubulin detyrosination in fibroblasts inhibits MT disassembly. This inhibition is relieved by overexpression of the depolymerizing motor mitotic centromere-associated kinesin (MCAK). Conversely, suppression of MCAK expression prevents disassembly of normal tyrosinated MTs in fibroblasts. Detyrosination of MTs suppresses the activity of MCAK in vitro, apparently as the result of a decreased affinity of the adenosine diphosphate (ADP)–inorganic phosphate- and ADP-bound forms of MCAK for the MT lattice. Detyrosination also impairs MT disassembly in neurons and inhibits the activity of the neuronal depolymerizing motor KIF2A in vitro. These results indicate that MT depolymerizing motors are directly inhibited by the detyrosination of tubulin, resulting in the stabilization of cellular MTs. Detyrosination of transiently stabilized MTs may give rise to persistent subpopulations of disassembly-resistant polymers to sustain subcellular cytoskeletal differentiation.     

Essential role of hyperacetylated microtubules in innate immunity escape orchestrated by the EBV-encoded BHRF1 protein

Innate immunity constitutes the first line of defense against viruses, in which mitochondria play an important role in the induction of the interferon (IFN) response. BHRF1, a multifunctional viral protein expressed during Epstein-Barr virus reactivation, modulates mitochondrial dynamics and disrupts the IFN signaling pathway. Mitochondria are mobile organelles that move through the cytoplasm thanks to the cytoskeleton and in particular the microtubule (MT) network. MTs undergo various post-translational modifications, among them tubulin acetylation. In this study, we demonstrated that BHRF1 induces MT hyperacetylation to escape innate immunity. Indeed, the expression of BHRF1 induces the clustering of shortened mitochondria next to the nucleus. This “mito-aggresome” is organized around the centrosome and its formation is MT-dependent. We also observed that the α-tubulin acetyltransferase ATAT1 interacts with BHRF1. Using ATAT1 knockdown or a non-acetylatable α-tubulin mutant, we demonstrated that this hyperacetylation is necessary for the mito-aggresome formation. Similar results were observed during EBV reactivation. We investigated the mechanism leading to the clustering of mitochondria, and we identified dyneins as motors that are required for mitochondrial clustering. Finally, we demonstrated that BHRF1 needs MT hyperacetylation to block the induction of the IFN response. Moreover, the loss of MT hyperacetylation blocks the localization of autophagosomes close to the mito-aggresome, impeding BHRF1 to initiate mitophagy, which is essential to inhibiting the signaling pathway. Therefore, our results reveal the role of the MT network, and its acetylation level, in the induction of a pro-viral mitophagy.     

Validity of ultrasound muscle thickness measurements for predicting leg skeletal muscle mass in healthy Japanese middle-aged and older individuals

Background

The skeletal muscle mass of the lower limb plays a role in its mobility during daily life. From the perspective of physical resources, leg muscle mass dominantly decreases after the end of the fifth decade. Therefore, an accurate estimate of the muscle mass is important for the middle-aged and older population. The present study aimed to clarify the validity of ultrasound muscle thickness (MT) measurements for predicting leg skeletal muscle mass (SM) in the healthy Japanese middle-aged and older population.

Findings

MTs at four sites of the lower limb and the bone-free lean tissue mass (LTM) of the right leg were determined using brightness-mode ultrasonography and dual-energy X-ray absorptiometry (DXA), respectively, in 44 women and 33 men, 52- to 78-years old. LTM was used as a representative variable of leg skeletal muscle mass. In the model-development group (30 women and 22 men), regression analysis produced an equation with R² and standard error of the estimate (SEE) of 0.958 and 0.3 kg, respectively: LTM (kg) = 0.01464 × (MT_SUM×L) (cm²) - 2.767, where MT_SUM is the sum of the product of MTs at four sites, and L is length of segment where MT is determined. The estimated LTM (7.0 ± 1.7 kg) did not significantly differ from the measured LTM (7.0 ± 1.7 kg), without a significant systematic error on a Bland-Altman plot. The application of this equation for the cross-validation group (14 women and 11 men) did not yield a significant difference between the measured (7.2 ± 1.6 kg) or estimated (7.2 ± 1.6 kg) LTM and systematic error.

Conclusion

The developed prediction equation may be useful for estimating the lean tissue mass of the lower extremity for the healthy Japanese middle-aged and older population.