A new interaction force decomposition maximizing compensating forces under physical work constraints

Decomposition of interaction forces in manipulation tasks has a long research tradition. Interaction forces are often split into robustness-reflective and accelerating forces. While this decomposition is typically performed for the synthesis of interaction forces to be applied for example in the context of robotic grasping, less attention has been paid to the analysis of measured, human interaction forces. Here we present a new decomposition approach for interaction force analysis. It extends the intuitive solution known in literature for the two finger grasp and combines it with a physically motivated bounding constraint, which allows the maximization of robustness reflective forces. Advantages of our approach are illustrated with an example and are compared to existing decomposition approaches. In contrast to existing approaches the new approach is not limited in the number of interaction points and incorporates forces which are physically possible only

HEATR2 Plays a Conserved Role in Assembly of the Ciliary Motile Apparatus

Cilia are highly conserved microtubule-based structures that perform a variety of sensory and motility functions during development and adult homeostasis. In humans, defects specifically affecting motile cilia lead to chronic airway infections, infertility and laterality defects in the genetically heterogeneous disorder Primary Ciliary Dyskinesia (PCD). Using the comparatively simple Drosophila system, in which mechanosensory neurons possess modified motile cilia, we employed a recently elucidated cilia transcriptional RFX-FOX code to identify novel PCD candidate genes. Here, we report characterization of CG31320/HEATR2, which plays a conserved critical role in forming the axonemal dynein arms required for ciliary motility in both flies and humans. Inner and outer arm dyneins are absent from axonemes of CG31320 mutant flies and from PCD individuals with a novel splice-acceptor HEATR2 mutation. Functional conservation of closely arranged RFX-FOX binding sites upstream of HEATR2 orthologues may drive higher cytoplasmic expression of HEATR2 during early motile ciliogenesis. Immunoprecipitation reveals HEATR2 interacts with DNAI2, but not HSP70 or HSP90, distinguishing it from the client/chaperone functions described for other cytoplasmic proteins required for dynein arm assembly such as DNAAF1-4. These data implicate CG31320/HEATR2 in a growing intracellular pre-assembly and transport network that is necessary to deliver functional dynein machinery to the ciliary compartment for integration into the motile axoneme

Cytoplasmic HEATR2 is required for the pre-assembly of axonemal dynein machinery necessary for motility.

<p>(A) Immunofluorescence for axonemal dynein heavy chain 5 (DNAH5: green) on respiratory cells from patients with <i>HEATR2</i> mutations compared to non-related control cells, shows loss of type 1 and type 2 DNAH5-positive staining although axonemes are still present (acetylated tubulin:red). Nuclei are stained with DAPI. (Scale bar: 10 µm). (B) Extracts prepared from control human terminally differentiated respiratory airway cultures (40 days ALI, Epithelyx) were subjected to immunoprecipitation (IP) with antibodies to HEATR2 (Proteintech) or control rabbit immunoglobulin G (GFP). Resulting immunocomplexes (IP: right) as well as dilutions of original extract (INPUT: left) were subjected to immunoblot analysis with antibodies to HEATR2 (Proteintech) or DNAI2 (Abnova). (See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004577#pgen.1004577.s008" target="_blank">Figure S8</a>). (C) No staining of axonemal dynein light intermediate chain 1 (DNALI1: green) is observed in patients with <i>HEATR2</i> mutations. No signal above background is detected in patient cells, in contrast to strong axonemal localization in non-related control cells. Nuclei are stained with DAPI. (Scale bar: 10 µm). (D) The DNALI1 orthologue in fly, <i>CG6971::mVenus</i> (green), fails to localize to ciliary axonemes (Ci: cilia, marked with square bracket) of Ch neurons (magenta: 22C10/Futsch) in <i>CG31320</i> knock-down larvae. (E) Schematic of dynamic role of HEATR2 in developing airway epithelial MMCs. Progenitor cells exit the cell cycle to commit to the MMC lineage with primary cilia. These cells express low levels of RFX3 (light red nuclei). Other upstream factors governing multiciliogenesis (MCN, MYB) induce centriole amplification as well as expression of FOXJ1 (bright green nuclei), required for centriole docking. High FOXJ1 and RFX3 drive a cilia motility transcriptional cascade leading to high expression of HEATR2 as well as expression of axonemal dynein components. HEATR2 is involved in the pre-assembly and/or delivery of future dynein arms to the apical cilia base. In fully mature MMCs, once inner and outer arm dynein complexes are delivered and incorporated into motile ciliary axonemes, relative levels of HEATR2 as well as RFX3 and FOXJ1 are reduced. This conserved regulatory motility module is required to drive high levels of HEATR2 when axonemal dyneins are being assembled and trafficked.</p

CCDC103 mutations cause primary ciliary dyskinesia by disrupting assembly of ciliary dynein arms

Cilia are essential for fertilization, respiratory clearance, cerebrospinal fluid circulation, and to establish laterality(1). Cilia motility defects cause Primary Ciliary Dyskinesia (PCD, MIM 242650), a disorder affecting 1:15-30,000 births. Cilia motility requires the assembly of multisubunit dynein arms that drive cilia bending(2). Despite progress in understanding the genetic basis of PCD, mutations remain to be identified for several PCD linked loci(3). Here we show that the zebrafish cilia paralysis mutant schmalhans(tn222) (smh) mutant encodes the coiled-coil domain containing 103 protein (Ccdc103), a foxj1a regulated gene. Screening 146 unrelated PCD families identified patients in six families with reduced outer dynein arms, carrying mutations in CCDC103. Dynein arm assembly in smh mutant zebrafish was rescued by wild-type but not mutant human CCDC103. Chlamydomonas Ccdc103 functions as a tightly bound, axoneme-associated protein. The results identify Ccdc103 as a novel dynein arm attachment factor that when mutated causes Primary Ciliary Dyskinesia

Cytoplasmic HEATR2 is expressed during early ciliogenesis.

<p>(A–C) Immunostaining of control human nasal brush epithelia reveals endogenous HEATR2 (red: Novus (A), Proteintech (C)) is highly enriched in the cytoplasm of developing MMC when components of outer dynein arms (B: DNAH5, red; C: DNAI2: green) as well as inner dynein arms (A–C: DNALI1, purple) are predominantly cytoplasmic. Arrowheads highlight fully mature MMCs where these components are exclusively axonemal and with relatively lower levels of HEATR2. Arrows highlight immature MMCs for comparison. Nuclei are stained with DAPI (blue). (Scale bar: A–C, 10 µm) (D) Double immunofluorescence of 22C10 (magenta: Futsch, cytoplasmic/membrane marker, but not cilium, of all sensory neurons) and <i>CG31320::mVenus</i> (green) indicates there is cytoplasmic but no ciliary localization of CG31320 in stage 16 Ch neurons (Ci: cilia, marked with square bracket). As this construct uses the upstream regulatory region of <i>CG31320</i> containing the X and Fox motifs to drive reporter expression, it further supports regulation occurs via these sites. (See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004577#pgen.1004577.s007" target="_blank">Figure S7</a>).</p

<i>CG31320</i>/<i>HEATR2</i> orthologues share conserved upstream regulatory FOX motifs and X-boxes of a master cilia motility transcriptional programme.

<p>(A) Using the human upstream epigenetic markings and conservation to mouse and rat to define conserved predicted regulatory elements, we focused analysis on the 500 bp upstream of the <i>HEATR2</i> ATG and syntenic regions in other species to identify X-box sequences, along with the nearest conserved FOX motifs. These sequences are coloured where they conform to recognized core consensus sequences for generic FOX proteins (RYMAAYA <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004577#pgen.1004577-Kaufmann1" target="_blank">[71]</a>) and RFX (RYYRYYN_(1–3)RRNRAC <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004577#pgen.1004577-Laurenon1" target="_blank">[42]</a>). Nucleotides are shown in grey if they vary from the consensus. Note for the second identified X-box site the 3′ site is extremely well-matched whilst the 5′ half-site is often more degenerate <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004577#pgen.1004577-ElZein1" target="_blank">[37]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004577#pgen.1004577-Newton1" target="_blank">[58]</a>. The distance from the Fox motif and X-box to the transcription start site is indicated, or else the distance to the ATG is indicated if a sizeable 5′UTR is present (<i>i.e. D. melanogaster, C. lupus</i>). An expanded table of the analysis is provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004577#pgen.1004577.s010" target="_blank">Table S2</a>. (B) ChIP-Seq data reveals a single, specific RFX3 peak 200 bp upstream from the transcriptional start site in OF1 mouse primary differentiated ependymal cell culture. Insert illustrates the two X-boxes bioinformatically predicted within the peak sequence. (C) Directed ChIP-qPCR data validates RFX3 occupancy is enriched at <i>Heatr2</i> promoter in OF1 cells, normalized to known target gene <i>Dyn2li1</i> and relative to a control sequence, downstream region in the <i>Dync2li1</i> gene. (D) <i>Heatr2</i> expression is ≈55% reduced in <i>Rfx3^−/−</i> ependymal cells similar to reductions in expression observed for two known direct Rfx3 targets, <i>Dync2li1</i> and <i>Bbs5</i>. qPCR data represent the average of three different assays performed in triplicate ± SEM. All data are considered significant using Student's t-test. (<i>Heatr2 P</i> = 0.003652632; <i>Dync2li P</i> = 0.013123897; <i>Bbs5 P</i> = 0.022511438).</p

<i>HEATR2</i> splice mutation results in alteration of the final conserved HEAT repeat and protein instability.

<p>(A) Pedigree of related families of UK-Pakistani descent. IV∶4 identifies the proband, also designated by the arrow. Solid symbols (individuals IV∶1, IV∶4 and IV∶10) indicate those affected with PCD. Double lines indicate consanguineous marriages. The individuals labeled DNA signified those that had their DNA included in SNP genotyping. (B) Schematic of <i>HEATR2</i> transcript showing the transversion mutation (<i>ENST00000297440:c.2432-1G>C</i>) affecting the splice acceptor site of the final exon. The mutation results in inactivation of this splice site and utilization of an adjacent downstream cryptic splice acceptor site in exon 13, causing a 2-nucleotide AG deletion in the <i>HEATR2</i> transcript, resulting in a frameshift in translation (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004577#pgen.1004577.s002" target="_blank">Figure S2B</a>). This is predicted to alter the final 44 amino acids of the protein and add an additional 33 amino acids with creation of a novel termination signal at codon 888 in the 3′UTR (See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004577#pgen.1004577.s003" target="_blank">Figure S3A</a>). This mutation disrupts the final highly conserved HEAT repeat and alters the C-terminus of the ARM-type fold superfamily domain (red). (C) Relative expression levels of <i>HEATR2</i> transcript by RT-qPCR, when normalized to the reference <i>TBP</i> gene. (D) The PCD transversion mutation (<i>ENST00000297440:c.2432-1G>C</i>) does not affect <i>HEATR2</i> transcript stability or gross splicing as shown by RT-PCR on parental control (C) and patient (P*) cDNA from LCLs. PCR products spanning the gene including the splice acceptor mutation at Exon 11–13 and Exon 12-3′UTR show no obvious alterations in size. Direct sequencing confirmed a 2 base pair deletion consistent with efficient splicing to the cryptic splice acceptor at the start of exon 13 in PCD patients (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004577#pgen.1004577.s002" target="_blank">Figure S2B</a>). (E) Western blot analysis on total protein extracts from unrelated control, heterozygous parental and homozygous patient LCLs demonstrates the PCD mutation (<i>ENST00000297440:c.2432-1G>C</i>) results in an elongated HEATR2 protein present at reduced levels implying instability. The slight shift in mobility of the protein in the patient is consistent with the predicted 3 kDa size shift due to the amino acid alterations described. β-actin is used as a loading control. (For longer exposure see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004577#pgen.1004577.s003" target="_blank">Figure S3B</a>). (F) Levels of HEATR2 protein normalized relative to β-actin reveal that parental samples which are heterozygous for the mutation shows a reduction to ≈50% of that of unrelated controls whilst the homozygous patient sample shows a reduction to ≈3% of control levels.</p

HEATR2 is highly expressed in tissues with motile cilia.

<p>(A) Developmental changes in gene expression were assayed by RT-qPCR on RNA extracted from wild-type mouse lungs and trachea from E14.5-P2 (N = 3 independent biological samples for each time-point). qPCR data represents the average of three different assays for three samples performed in triplicate ± SEM. Kruskal-Wallis non-parametric analysis of variance was performed and was significant for all genes (<i>Zmynd10 P</i> = 1.558e⁻⁰⁸; <i>Dnahc5 P</i> = 1.141e⁻⁰⁵; <i>Dnali1 P</i> = 9.814e⁻⁰⁶; <i>Foxj1 P</i> = 1.956e⁻⁰⁹; <i>Heatr2 P</i> = 1.604e⁻⁰⁶; <i>Rfx3 P</i> = 6.68e⁻⁰⁶). (B,C) Immunostaining on sections of E15.5 mouse lungs, where strong RFX3 and FOXJ1 signals are co-expressed in a “salt and pepper” pattern only in large proximal airways, not smaller, more distal airways (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004577#pgen.1004577.s006" target="_blank">Figure S6A,B</a>). Although they are not yet multiciliated, these cells also express components of axonemal dyneins and high levels of HEATR2 in their cytoplasm. (Scale bar: B,C = 50 µm, B′,C′ = 10 µm). (D–G) Immunostaining of human nasal brush epithelial cells for: (D) RFX3 (HPA: red), acetylated tubulin (green) and DNALI1 (SC: purple); (E) HEATR2 (Novus: red), FOXJ1 (green) and DNALI1 (SC: purple); and (F,G) HEATR2 (Proteintech: red), FOXJ1 (green) and RFX3 (SC: purple). White arrowheads highlight fully mature motile, multiciliated cells (MMCs) that express lower nuclear RFX3 and FOXJ1 with reduced HEATR2 and with axonemal dynein components entirely in cilia. Arrows highlight immature MMCs for comparison. HEATR2 is entirely cytoplasmic at all stages examined (Scale bar: D–G = 10 µm).</p

Frequency of visible dynein arms in <i>Drosophila</i> sensory-neural specific <i>CG31320</i> RNAi mutant Ch neuronal axonemes by TEM.

<p><b><u>Key:</u></b></p><p>n: number of axonemal microtubule doublets scored. Visible dynein arm: the percentage (number) of axonemal microtubule doublets for which staining consistent with a dynein arm is visible. In both instances, Fisher's Exact Test shows significant reduction in the knock-down cilia (<i>P</i>≤0.0001).</p><p>Frequency of visible dynein arms in <i>Drosophila</i> sensory-neural specific <i>CG31320</i> RNAi mutant Ch neuronal axonemes by TEM.</p