A Cilia Independent Role of Ift88/Polaris during Cell Migration

Boehlke, Christopher; Janusch, Heike; Hamann, Christoph; Powelske, Christian; Mergen, Miriam; Herbst, Henriette; Kotsis, Fruzsina; Nitschke, Roland; Kuehn, E. Wolfgang

doi:10.1371/journal.pone.0140378

Cited by 47 publications

(36 citation statements)

References 45 publications

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“…The meaning behind this correlation remains unexplored but it does support the hypothesis that Ift88 is more tightly connected to ciliogenesis. Although IFT88 deletion is perhaps a more appropriate mechanism to specifically study the cilium, particularly in the context of skeletogenesis, Ift88 is known to have a cilium‐independent role in cell migration . For growth plate studies, however, this is less offensive than Kif3a's independent role in Wnt signaling.…”

Section: Disrupting Primary Cilia Results In Abnormal Growth Plate Momentioning

confidence: 99%

“…Although IFT88 deletion is perhaps a more appropriate mechanism to specifically study the cilium, particularly in the context of skeletogenesis, Ift88 is known to have a cilium-independent role in cell migration. 150 For growth plate studies, however, this is less offensive Overall, we recommend that researchers thoughtfully select their mechanism of cilium disruption and be cognizant of potential non-ciliary effects that could influence chondrocyte behavior and skeletal phenotype.…”

Section: The Mechanism For Primary Cilium Disruption Is An Important mentioning

confidence: 99%

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The primary cilium as a signaling nexus for growth plate function and subsequent skeletal development

Moore

Jacobs

2017

Journal Orthopaedic Research

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The primary cilium is a solitary, antenna-like sensory organelle with many important roles in cartilage and bone development, maintenance, and function. The primary cilium's potential role as a signaling nexus in the growth plate makes it an attractive therapeutic target for diseases and disorders associated with bone development and maintenance. Many signaling pathways that are mediated by the cilium-such as Hh, Wnt, Ihh/PTHrP, TGFβ, BMP, FGF, and Notch-are also known to influence endochondral ossification, primarily by directing growth plate formation and chondrocyte behavior. Although a few studies have demonstrated that these signaling pathways can be directly tied to the primary cilium, many pathways have yet to be evaluated in context of the cilium. This review serves to bridge this knowledge gap in the literature, as well as discuss the cilium's importance in the growth plate's ability to sense and respond to chemical and mechanical stimuli. Furthermore, we explore the importance of using the appropriate mechanism to study the cilium in vivo and suggest IFT88 deletion is the best available technique. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:533-545, 2018.

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Section: Disrupting Primary Cilia Results In Abnormal Growth Plate Momentioning

confidence: 99%

Section: The Mechanism For Primary Cilium Disruption Is An Important mentioning

confidence: 99%

The primary cilium as a signaling nexus for growth plate function and subsequent skeletal development

Moore

Jacobs

2017

Journal Orthopaedic Research

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“…This has also previously been associated with the regulation of multiple chondrocyte responses (37–39, 47–49), most of which are proposed to be indicative of roles for the cilium. Importantly, however, IFT88, and indeed this mutation, are both associated with cell biology that is not proven to be attributable to cilia function—for example, migration (50), cell division (51), and planar cell polarity (52). …”

Section: Discussionmentioning

confidence: 99%

Cilia protein IFT88 regulates extracellular protease activity by optimizing LRP‐1–mediated endocytosis

et al. 2018

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Matrix protease activity is fundamental to developmental tissue patterning and remains influential in adult homeostasis. In cartilage, the principal matrix proteoglycan is aggrecan, the protease-mediated catabolism of which defines arthritis; however, the pathophysiologic mechanisms that drive aberrant aggrecanolytic activity remain unclear. Human ciliopathies exhibit altered matrix, which has been proposed to be the result of dysregulated hedgehog signaling that is tuned within the primary cilium. Here, we report that disruption of intraflagellar transport protein 88 (IFT88), a core ciliary trafficking protein, increases chondrocyte aggrecanase activity in vitro. We find that the receptor for protease endocytosis in chondrocytes, LDL receptor–related protein 1 (LRP-1), is unevenly distributed over the cell membrane, often concentrated at the site of cilia assembly. Hypomorphic mutation of IFT88 disturbs this apparent hot spot for protease uptake, increases receptor shedding, and results in a reduced rate of protease clearance from the extracellular space. We propose that IFT88 and/or the cilium regulates the extracellular remodeling of matrix—independently of Hedgehog regulation—by enabling rapid LRP-1–mediated endocytosis of proteases, potentially by supporting the creation of a ciliary pocket. This result highlights new roles for the cilium’s machinery in matrix turnover and LRP-1 function, with potential relevance in a range of diseases.—Coveney, C. R., Collins, I., Mc Fie, M., Chanalaris, A., Yamamoto, K., Wann, A. K. T. Cilia protein IFT88 regulates extracellular protease activity by optimizing LRP-1–mediated endocytosis.

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“…It is becoming increasingly clear that cilia proteins can also have extraciliary localizations and functions [18, 26, 37, 38]. Further work is needed to determine which ciliary proteins have extraciliary functions, and whether these extraciliary functions of cilia proteins contribute to the pathology of ciliopathies.…”

Section: Discussionmentioning

confidence: 99%

Fixation methods can differentially affect ciliary protein immunolabeling

Hua

Ferland

2017

Cilia

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BackgroundPrimary cilia are immotile, microtubule-based organelles present on most cells. Defects in primary cilia presence/function result in a category of developmental diseases referred to as ciliopathies. As the cilia field progresses, there is a need to consider both the ciliary and extraciliary roles of cilia proteins. However, traditional fixation methods are not always suitable for examining the full range of localizations of cilia proteins. Here, we tested a variety of fixation methods with commonly used cilia markers to determine the most appropriate fixation method for different cilia proteins.MethodsMouse inner medullary collecting duct and human retinal pigmented epithelial cells were grown to confluence, serum starved, and fixed with one of the following fixation agents: paraformaldehyde–sucrose, paraformaldehyde–PBS, methanol, cytoskeletal buffer followed by methanol, or three variations of cytoskeletal buffer–paraformaldehyde fixation. Each cell type and fixation method combination was probed with the following ciliary markers: acetylated α-tubulin, detyrosinated tubulin, polyglutamylated tubulin, β-tubulin, adenylyl cyclase 3 (AC3), ADP-ribosylation factor-like protein 13b (Arl13b), centrosome and spindle pole associated protein 1 (CSPP1), or intraflagellar transport protein 20 (IFT20). Intraflagellar transport protein 88 (IFT88) and GM130 (Golgi marker) were also used. We assessed actin (via phalloidin) and microtubule integrity, centrioles, cilia, and two extraciliary sites (mitotic figures and Golgi).ResultsFor the cilia markers examined, paraformaldehyde fixation preserved cilia immunolabeling of cilia-membrane proteins (AC3 and Arl13b), but failed to reveal cilia immunostaining of axonemal proteins (CSPP1 and IFT20). Methanol revealed cilia labeling for some axonemal proteins, but not others, and this depended on cell type. Generally, any method that first included a wash in cytoskeletal buffer, before fixing, revealed more distinct cilia immunolabeling for axonemal proteins (CSPP1, IFT20, and IFT88), but resulted in the loss of cilia labeling for cilia-membrane proteins (AC3 and Arl13b). All three different post-translational modifications of tubulin antibodies positively immunolabeled cilia in all fixation methods tested. Ultimately, we found that fixing cells in a solution of paraformaldehyde prepared in cytoskeletal buffer allowed for the preservation of cilia immunolabeling for most cilia proteins tested and allowed visualization of two extraciliary sites (mitotic figures and Golgi).ConclusionSome general patterns were observed to guide in the choice of a fixation agent. Cilia-membrane proteins generally benefit from quick fixation with no prior permeabilization, whereas axonemal proteins tend to benefit from permeabilization and use of cytoskeletal buffer.

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A Cilia Independent Role of Ift88/Polaris during Cell Migration

Cited by 47 publications

References 45 publications

The primary cilium as a signaling nexus for growth plate function and subsequent skeletal development

The primary cilium as a signaling nexus for growth plate function and subsequent skeletal development

Cilia protein IFT88 regulates extracellular protease activity by optimizing LRP‐1–mediated endocytosis

Fixation methods can differentially affect ciliary protein immunolabeling

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