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Papers by ryan gray

Most motile cilia have a stereotyped structure of nine microtubule outer doublets and a single ce... more Most motile cilia have a stereotyped structure of nine microtubule outer doublets and a single central pair of microtubules. The central pair microtubules are surrounded by a set of proteins, termed the central pair apparatus. A specific kinesin, Klp1 projects from the central pair and contributes to ciliary motility in Chlamydomonas. The vertebrate orthologue, Kif9 is required for beating in mouse sperm flagella, but the mechanism of Kif9/Klp1 function remains poorly defined. Here, using Xenopus epidermal multiciliated cells, we show that Kif9 is necessary for ciliary motility as well as leads to defects in the distal localization of not only central pair proteins, but also radial spokes and dynein arms. In addition, single-molecule assays in vitro revealed that Xenopus Kif9 is a processive motor, though like axonemal dyneins it displays no processivity in ciliary axonemes in vivo. Thus, our data suggest that Kif9 plays both indirect and direct role in ciliary motility.

Aging, Neuropsychology, and Cognition, 2019

We thank Dr Eleftheria Vaportzis for her input during the early development of the study.

PLOS Genetics, 2018

Cerebrospinal fluid flow is crucial for neurodevelopment and homeostasis of the ventricular syste... more Cerebrospinal fluid flow is crucial for neurodevelopment and homeostasis of the ventricular system of the brain, with localized flow being established by the polarized beating of the ependymal cell (EC) cilia. Here, we report a homozygous one base-pair deletion, c.1193delT (p.Leu398Glnfs*2), in the Kinesin Family Member 6 (KIF6) gene in a child displaying neurodevelopmental defects and intellectual disability. To test the pathogenicity of this novel human KIF6 mutation we engineered an analogous C-terminal truncating mutation in mouse. These mutant mice display severe, postnatal-onset hydrocephalus. We generated a Kif6-LacZ transgenic mouse strain and report expression specifically and uniquely within the ependymal cells (ECs) of the brain, without labeling other multiciliated mouse tissues. Analysis of Kif6 mutant mice with scanning electron microscopy (SEM) and immunofluorescence (IF) revealed specific defects in the formation of EC cilia, without obvious effect of cilia of other multiciliated tissues. Dilation of the ventricular system and defects in the formation of EC cilia were also observed in adult kif6 mutant zebrafish. Finally, we report Kif6-GFP localization at the axoneme and basal bodies of multi-ciliated cells (MCCs) of the mucociliary Xenopus epidermis. Overall, this work describes the first clinically-defined KIF6 homozygous null mutation in human and defines KIF6 as a conserved mediator of neurological development with a specific role for EC ciliogenesis in vertebrates.

Cold Spring Harbor protocols, 2010

Embryos of the frog Xenopus laevis are an ideal model system for in vivo imaging of dynamic biolo... more Embryos of the frog Xenopus laevis are an ideal model system for in vivo imaging of dynamic biological processes, from the inner workings of individual cells to the reshaping of tissues during embryogenesis. Their externally developing embryos are more amenable to in vivo analysis than internally developing mammalian embryos, and the large size of the embryos make them particularly suitable for time-lapse analysis of tissue-level morphogenetic events. In addition, individual cells in Xenopus embryos are larger than those in other vertebrate models, making them ideal for imaging cell behavior and subcellular processes (e.g., following the dynamics of fluorescent fusion proteins in living or fixed cells and tissues). Xenopus embryos are amenable to simple manipulations of gene function, including knockdown and misexpression, and the large number of embryos available allows even an inexperienced researcher to perform hundreds of such manipulations per day. Transgenesis is quite effecti...

CSH protocols, 2008

INTRODUCTIONImmunocytochemistry (ICC) is widely exploited in studying mammalian systems, but is u... more INTRODUCTIONImmunocytochemistry (ICC) is widely exploited in studying mammalian systems, but is underutilized among Xenopus developmental biologists. This stems, in part, from the relatively small number of Xenopus antibodies available for use in research. Common misconceptions about ICC in Xenopus embryos also prevail, discouraging researchers from trying the procedure. However, ICC with Xenopus is simple and effective. This article describes methods for whole-mount ICC in Xenopus embryos. Also included are simple procedures to quench autofluorescence of Xenopus and to remove surface pigment from embryos which may interfere with fluorescence imaging. The methods described here are useful for detecting tissue-specific probes (e.g., 12/101 to detect somites). They are also effective for imaging the cytoskeleton (e.g., α-tubulin to detect microtubules) or localizing specific proteins at the subcellular level (e.g., ZO-1 to detect tight junctions). In addition, combining ICC with in si...

Science, 2010

Form and Function The Planar Cell Polarity (PCP) signaling pathway governs cell movements that dr... more Form and Function The Planar Cell Polarity (PCP) signaling pathway governs cell movements that drive axis elongation and neural tube closure in vertebrate embryos, and certain vertebrate PCP proteins have also been implicated in ciliogenesis. Likewise, the septin cytoskeleton controls diverse cell behaviors, such as cytokinesis and cell migration, but little is known about how septin functions are regulated in vivo. Kim et al. (p. 1337 , published online 29 July; see the Perspective by Barral ) found that control of septins by the PCP effector protein, Fritz, was a crucial control point for morphogenesis and ciliogenesis. During neural tube closure, Fritz-mediated septin localization maintained cell shape but not cell polarity. In ciliated epithelial cells, Fritz was required for assembly of the septin rings at the base of cilia, which are needed for normal ciliogenesis and signaling.

Proceedings of the National Academy of Sciences, 2012

Breast cancer progression involves genetic changes and changes in the extracellular matrix (ECM).... more Breast cancer progression involves genetic changes and changes in the extracellular matrix (ECM). To test the importance of the ECM in tumor cell dissemination, we cultured epithelium from primary human breast carcinomas in different ECM gels. We used basement membrane gels to model the normal microenvironment and collagen I to model the stromal ECM. In basement membrane gels, malignant epithelium either was indolent or grew collectively, without protrusions. In collagen I, epithelium from the same tumor invaded with protrusions and disseminated cells. Importantly, collagen I induced a similar initial response of protrusions and dissemination in both normal and malignant mammary epithelium. However, dissemination of normal cells into collagen I was transient and ceased as laminin 111 localized to the basal surface, whereas dissemination of carcinoma cells was sustained throughout culture, and laminin 111 was not detected. Despite the large impact of ECM on migration strategy, transc...

Mechanisms of Development, 2009

Developmental Dynamics, 2009

Dishevelled (Dvl) proteins are key transducers of Wnt signaling encoded by members of a multigene... more Dishevelled (Dvl) proteins are key transducers of Wnt signaling encoded by members of a multigene family in vertebrates. We report here the divergent, tissue-specific expression patterns for all three Dvl genes in Xenopus embryos, which contrast dramatically with their expression patterns in mice. Moreover, we find that the expression patterns of Dvl genes in the chick diverge significantly from those of Xenopus. In addition, in hemichordates, an outgroup to chordates, we find that the one Dvl gene is dynamically expressed in a tissue-specific manner. Using knockdowns, we find that Dvl1 and Dvl2 are required for early neural crest specification and for somite segmentation in Xenopus. Most strikingly, we report a novel role for Dvl3 in the maintenance of gene expression in muscle and in the development of the Xenopus sclerotome. These data demonstrate that the expression patterns and developmental functions of specific Dvl genes have diverged significantly during chordate evolution.

Biomaterials, 2010

Non-viral gene delivery vectors were developed for efficient gene transfer to hard to transfect m... more Non-viral gene delivery vectors were developed for efficient gene transfer to hard to transfect mouse mammary epithelial cells. Ten modified versions of the same base poly(beta-amino ester), poly(1,4-butanediol diacrylate-co-5-amino-1-pentanol), were tested in both traditional 2-D monolayer and in 3-D organotypic cultures. The polymers self-assembled with plasmid DNA encoding enhanced green fluorescent protein to form nanoparticles (~100 nm) used to transfect the cells. Nanoparticle transfection efficacy was tuned by changes in synthesis and fabrication conditions and the transfection efficacy was analyzed using confocal microscopy and flow cytometry. The best performing polymeric nanoparticles transfected 57±6% of the cells in 2-D culture and 6±1% of the cells in 3-D culture. Small modifications to the polymer end-capping molecules and tuning of polymer molecular weight could either significantly enhance the transfection efficacy up to 6-fold or instead abolish efficacy completely. The efficacy of leading polymers was higher than that of the commercial transfection agent FuGENE® HD by a factor of 13 in 2-D and 2 in 3-D. These non-viral nanoparticles may be useful as delivery reagents or targeted therapeutics for breast cancer. This gene delivery strategy is also a promising approach for studying the normal development of the mammary gland.

Frontiers in Aging Neuroscience

During the normal course of aging, perception of speech-on-speech or “cocktail party” speech and ... more During the normal course of aging, perception of speech-on-speech or “cocktail party” speech and use of working memory (WM) abilities change. Musical training, which is a complex activity that integrates multiple sensory modalities and higher-order cognitive functions, reportedly benefits both WM performance and speech-on-speech perception in older adults. This mini-review explores the relationship between musical training, WM and speech-on-speech perception in older age (> 65 years) through the lens of the Ease of Language Understanding (ELU) model. Linking neural-oscillation literature associating speech-on-speech perception and WM with alpha-theta oscillatory activity, we propose that two stages of speech-on-speech processing in the ELU are underpinned by WM-related alpha-theta oscillatory activity, and that effects of musical training on speech-on-speech perception may be reflected in these frequency bands among older adults.