Multimodal nonlinear optical microscopy reveals critical role of kinesin-1 in cartilage development

He, Sicong; Xue, Wenqian; Duan, Zhigang; Sun, Qiqi; Li, Xuesong; Gan, H; Huang, Jiandong; Qu, Jianan Y.

doi:10.1364/boe.8.001771

Cited by 8 publications

(5 citation statements)

References 30 publications

(34 reference statements)

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“…This phenomenon was reported as an intracellular collagen fibril formation in a study of Kif5b-depleted mutant mice cartilage. 29 However, to the best of our knowledge, this type of collagen formation has not been reported in normal chondrocytes of articular cartilage. It is unknown whether this is a pathological or a normal physiological process.…”

Section: Discussionmentioning

confidence: 80%

Nonlabeling and quantitative assessment of chondrocyte viability in articular cartilage with intrinsic nonlinear optical signatures

Chen

Watkins

et al. 2020

Exp Biol Med (Maywood)

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Chondrocyte viability is a crucial factor for evaluating cartilage health. Most prevalent cell viability assays rely on dyes and are not applicable for in vivo or longitudinal studies. Here we demonstrated that the two-photon excited autofluorescence and second harmonic generation microscopy provided high-resolution imaging of cartilage tissue and distinguished live/dead chondrocytes by visual assessment. Furthermore, the normalized autofluorescence ratio was proposed as a quantitative indicator to determine chondrocyte viability. Based on the indicator, a curve fitting and simulated receiver operating characteristic method was proposed to identify the live/dead cell populations as well as the indicator threshold without dye labeling. Thus, it established the label-free imaging method for chondrocyte viability assay in cartilage tissue. Impact statement Chondrocytes are the only cellular component found in the cartilage, playing a critical role in maintaining the homeostasis of articular cartilage. The viability of chondrocytes is a crucial factor for evaluating cartilage health. However, the current prevalent cell viability assays rely on dye staining and thereby are not applicable in vivo or in longitudinal assessments. In this study, we demonstrate that the intrinsic signals such as two-photon excited autofluorescence and second harmonic generation can be used to classify live and dead chondrocytes in cartilage tissue. A quantitative measure is also proposed allowing development of automated assessment algorithms. The nonlabeling nature of this method suggests the potential applicability to nondestructive and in vivo assessment of cartilage health.

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Section: Discussionmentioning

confidence: 80%

Nonlabeling and quantitative assessment of chondrocyte viability in articular cartilage with intrinsic nonlinear optical signatures

Chen

Watkins

et al. 2020

Exp Biol Med (Maywood)

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“…KIF5B may have some cytokinesis independent roles in the chondrocytes. We have reported that the secretion of extracellular matrix proteins was affected in the mutant [14]. We also observed that the disruption of terminal differentiation of the chondrocytes and cell death in the mutant growth plate, which may not be directly related to the cytokinetic defect.…”

Section: Discussionmentioning

confidence: 88%

KIF5B modulates central spindle organization in late-stage cytokinesis in chondrocytes

et al. 2019

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Background The growth plate is a special region of the cartilage that drives longitudinal growth of long bones. Proliferating chondrocytes in the growth plate, arranged in columns, divide perpendicular to the long axis of the growth plate then intercalate to re-align with parental columns. Which molecular partners maintain growth plate columnar structures and chondrocyte cytokinesis has not been fully revealed. It is reported that kinesin family member 3A (KIF3A), a subunit of kinesin-2, plays an important role in maintaining columnar organization in growth plates via controlling primary cilia formation and cell proliferation. Result Here we identify kinesin family member 5B (KIF5B), the heavy chain of kinesin-1, a ubiquitously expressed motor protein for anterograde intracellular transport along the microtubule network, as a key modulator of cytokinesis in chondrocytes via maintenance of central spindle organization. We show that KIF5B is concentrated in the central spindle during cytokinesis in both primary chondrocytes and chondrogenic ATDC5 cells. Conclusion The failure of cytokinesis in KIF5B null chondrocytes leads to incomplete cell rotation, disrupting proliferation and differentiation, and results in a disorganized growth plate.

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“…Vesicular trafficking defects have been implicated in low bone mass phenotypes and bone fragility that are at least partly attributed to failure in collagen secretion [48][49][50]. Interestingly, impaired secretion of type II collagen has been demonstrated in Kif5b-deficient chondrocytes [14,51]. In proband 1 fibroblasts, type I collagen electrophoresis did not suggest a qualitative or quantitative collagen defect (S1 Fig) . More research is needed to determine whether impaired collagen secretion is contributing to the phenotype in KIF5B-related osteogenesis imperfecta.…”

Section: (Fig 1d)mentioning

confidence: 99%

Dominant negative variants in KIF5B cause osteogenesis imperfecta via down regulation of mTOR signaling

Marom,

Zhang,

Washington

et al. 2023

PLoS Genet

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Background Kinesin motor proteins transport intracellular cargo, including mRNA, proteins, and organelles. Pathogenic variants in kinesin-related genes have been implicated in neurodevelopmental disorders and skeletal dysplasias. We identified de novo, heterozygous variants in KIF5B, encoding a kinesin-1 subunit, in four individuals with osteogenesis imperfecta. The variants cluster within the highly conserved kinesin motor domain and are predicted to interfere with nucleotide binding, although the mechanistic consequences on cell signaling and function are unknown. Methods To understand the in vivo genetic mechanism of KIF5B variants, we modeled the p.Thr87Ile variant that was found in two patients in the C. elegans ortholog, unc-116, at the corresponding position (Thr90Ile) by CRISPR/Cas9 editing and performed functional analysis. Next, we studied the cellular and molecular consequences of the recurrent p.Thr87Ile variant by microscopy, RNA and protein analysis in NIH3T3 cells, primary human fibroblasts and bone biopsy. Results C. elegans heterozygous for the unc-116 Thr90Ile variant displayed abnormal body length and motility phenotypes that were suppressed by additional copies of the wild type allele, consistent with a dominant negative mechanism. Time-lapse imaging of GFP-tagged mitochondria showed defective mitochondria transport in unc-116 Thr90Ile neurons providing strong evidence for disrupted kinesin motor function. Microscopy studies in human cells showed dilated endoplasmic reticulum, multiple intracellular vacuoles, and abnormal distribution of the Golgi complex, supporting an intracellular trafficking defect. RNA sequencing, proteomic analysis, and bone immunohistochemistry demonstrated down regulation of the mTOR signaling pathway that was partially rescued with leucine supplementation in patient cells. Conclusion We report dominant negative variants in the KIF5B kinesin motor domain in individuals with osteogenesis imperfecta. This study expands the spectrum of kinesin-related disorders and identifies dysregulated signaling targets for KIF5B in skeletal development.

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Multimodal nonlinear optical microscopy reveals critical role of kinesin-1 in cartilage development

Cited by 8 publications

References 30 publications

Nonlabeling and quantitative assessment of chondrocyte viability in articular cartilage with intrinsic nonlinear optical signatures

Nonlabeling and quantitative assessment of chondrocyte viability in articular cartilage with intrinsic nonlinear optical signatures

KIF5B modulates central spindle organization in late-stage cytokinesis in chondrocytes

Dominant negative variants in KIF5B cause osteogenesis imperfecta via down regulation of mTOR signaling

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