scholarly journals Acute inhibition of heterotrimeric kinesin-2 function reveals mechanisms of intraflagellar transport in mammalian cilia

2018 ◽  
Author(s):  
Martin F. Engelke ◽  
Bridget Waas ◽  
Sarah E. Kearns ◽  
Ayana Suber ◽  
Allison Boss ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Primary Cilia ◽  
Intraflagellar Transport ◽  
Genetic Approach ◽  
Ciliated Cells ◽  
Loss Of Function ◽  
Double Knockout ◽  
Chemical Genetic ◽  
Species Specific ◽  
And Function

ABSTRACTThe trafficking of components within cilia, called intraflagellar transport (IFT), is powered by kinesin-2 and dynein-2 motors. Loss of function in any subunit of the heterotrimeric KIF3A/KIF3B/KAP kinesin-2 motor prevents ciliogenesis in mammalian cells and has hindered an understanding of how kinesin-2 motors function in IFT. We used a chemical-genetic approach to engineer an inhibitable KIF3A/KIF3B (i3A/i3B) kinesin-2 motor that is capable of rescuing WT motor function in Kif3a/Kif3b double-knockout cells. Inhibitor addition blocks ciliogenesis or, if added to ciliated cells, blocks IFT within two minutes, which leads to a complete loss of primary cilia within six hours. The kinesin-2 family members KIF3A/KIF3C and KIF17 cannot rescue ciliogenesis in Kif3a/Kif3b double-knockout cells nor delay the disassembly of full-formed cilia upon i3A/i3B inhibition. These data suggest that KIF3A/KIF3B/KAP is the sole and essential motor for cilia assembly and function in mammalian cells, indicating a species-specific adaptation of kinesin-2 motors for IFT function.

scholarly journals Dynein-2 intermediate chains play crucial but distinct roles in primary cilia formation and function

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Laura Vuolo ◽  
Nicola L Stevenson ◽  
Kate J Heesom ◽  
David J Stephens
Keyword(s):  
Transition Zone ◽  
Primary Cilia ◽  
Complex Function ◽  
Intraflagellar Transport ◽  
Loss Of Function ◽  
Jeune Syndrome ◽  
Cilia Formation ◽  
Microtubule Motor ◽  
And Function ◽  
Intermediate Chains

The dynein-2 microtubule motor is the retrograde motor for intraflagellar transport. Mutations in dynein-2 components cause skeletal ciliopathies, notably Jeune syndrome. Dynein-2 contains a heterodimer of two non-identical intermediate chains, WDR34 and WDR60. Here, we use knockout cell lines to demonstrate that each intermediate chain has a distinct role in cilium function. Using quantitative proteomics, we show that WDR34 KO cells can assemble a dynein-2 motor complex that binds IFT proteins yet fails to extend an axoneme, indicating complex function is stalled. In contrast, WDR60 KO cells do extend axonemes but show reduced assembly of dynein-2 and binding to IFT proteins. Both proteins are required to maintain a functional transition zone and for efficient bidirectional intraflagellar transport. Our results indicate that the subunit asymmetry within the dynein-2 complex is matched with a functional asymmetry between the dynein-2 intermediate chains. Furthermore, this work reveals that loss of function of dynein-2 leads to defects in transition zone architecture, as well as intraflagellar transport.

scholarly journals Transient accumulation and bidirectional movement of KIF13B in primary cilia

2021 ◽  
Author(s):  
Alice Dupont Juhl ◽  
Zeinab Anvarian ◽  
Julia Berges ◽  
Daniel Wustner ◽  
Lotte B Pedersen
Keyword(s):  
Mammalian Cells ◽  
Live Cell Imaging ◽  
Primary Cilia ◽  
Cell Imaging ◽  
Intraflagellar Transport ◽  
Cytoplasmic Dynein ◽  
Ciliary Membrane ◽  
Bidirectional Movement ◽  
And Function ◽  
Male Specific

Primary cilia are microtubule-based sensory organelles whose assembly and function rely on the conserved bidirectional intraflagellar transport (IFT) system, which is powered by anterograde kinesin-2 and retrograde cytoplasmic dynein 2 motors. Nematodes additionally employ a male-specific kinesin-3 motor, KLP-6, which regulates ciliary content and function by promoting release of bioactive extracellular vesicles (EVs) from cilia. Here we show by live cell imaging that a KLP-6 homolog, KIF13B, undergoes bursts of bidirectional movement within primary cilia of cultured mammalian cells at 0.64 +/- 0.07 μm/s in the anterograde direction and at 0.39 +/- 0.06 μm/s in the retrograde direction, reminiscent of conventional IFT. In addition, we found that KIF13B undergoes EV-like release from the ciliary tip whereas a ciliary membrane marker, SMO-tRFP, remains stably associated with cilia during such EV release. Our results suggest that KIF13B, similar to KLP-6, regulates ciliary membrane content by promoting ciliary EV release, possibly in coordination with conventional IFT.

scholarly journals Codon harmonization reduces amino acid misincorporation in bacterially expressed P. falciparum proteins and improves their immunogenicity

AMB Express ◽  
2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Neeraja Punde ◽  
Jennifer Kooken ◽  
Dagmar Leary ◽  
Patricia M. Legler ◽  
Evelina Angov
Keyword(s):  
Protein Structure ◽  
Amino Acid ◽  
Codon Usage ◽  
Dna Sequences ◽  
Structural Integrity ◽  
Host Cells ◽  
Loss Of Function ◽  
Species Specific ◽  
And Function ◽  
The Impact

Abstract Codon usage frequency influences protein structure and function. The frequency with which codons are used potentially impacts primary, secondary and tertiary protein structure. Poor expression, loss of function, insolubility, or truncation can result from species-specific differences in codon usage. “Codon harmonization” more closely aligns native codon usage frequencies with those of the expression host particularly within putative inter-domain segments where slower rates of translation may play a role in protein folding. Heterologous expression of Plasmodium falciparum genes in Escherichia coli has been a challenge due to their AT-rich codon bias and the highly repetitive DNA sequences. Here, codon harmonization was applied to the malarial antigen, CelTOS (Cell-traversal protein for ookinetes and sporozoites). CelTOS is a highly conserved P. falciparum protein involved in cellular traversal through mosquito and vertebrate host cells. It reversibly refolds after thermal denaturation making it a desirable malarial vaccine candidate. Protein expressed in E. coli from a codon harmonized sequence of P. falciparum CelTOS (CH-PfCelTOS) was compared with protein expressed from the native codon sequence (N-PfCelTOS) to assess the impact of codon usage on protein expression levels, solubility, yield, stability, structural integrity, recognition with CelTOS-specific mAbs and immunogenicity in mice. While the translated proteins were expected to be identical, the translated products produced from the codon-harmonized sequence differed in helical content and showed a smaller distribution of polypeptides in mass spectra indicating lower heterogeneity of the codon harmonized version and fewer amino acid misincorporations. Substitutions of hydrophobic-to-hydrophobic amino acid were observed more commonly than any other. CH-PfCelTOS induced significantly higher antibody levels compared with N-PfCelTOS; however, no significant differences in either IFN-γ or IL-4 cellular responses were detected between the two antigens.

scholarly journals Prominins control ciliary length throughout the animal kingdom: New lessons from human prominin-1 and zebrafish prominin-3

2020 ◽  
Vol 295 (18) ◽  
pp. 6007-6022 ◽  
Author(s):  
József Jászai ◽  
Kristina Thamm ◽  
Jana Karbanová ◽  
Peggy Janich ◽  
Christine A. Fargeas ◽  
...  
Keyword(s):  
Primary Cilia ◽  
Photoreceptor Cells ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Loss Of Function ◽  
Animal Kingdom ◽  
Left Right Asymmetry ◽  
And Function ◽  
The Impact

Prominins (proms) are transmembrane glycoproteins conserved throughout the animal kingdom. They are associated with plasma membrane protrusions, such as primary cilia, as well as extracellular vesicles derived thereof. Primary cilia host numerous signaling pathways affected in diseases known as ciliopathies. Human PROM1 (CD133) is detected in both somatic and cancer stem cells and is also expressed in terminally differentiated epithelial and photoreceptor cells. Genetic mutations in the PROM1 gene result in retinal degeneration by impairing the proper formation of the outer segment of photoreceptors, a modified cilium. Here, we investigated the impact of proms on two distinct examples of ciliogenesis. First, we demonstrate that the overexpression of a dominant-negative mutant variant of human PROM1 (i.e. mutation Y819F/Y828F) significantly decreases ciliary length in Madin–Darby canine kidney cells. These results contrast strongly to the previously observed enhancing effect of WT PROM1 on ciliary length. Mechanistically, the mutation impeded the interaction of PROM1 with ADP-ribosylation factor–like protein 13B, a key regulator of ciliary length. Second, we observed that in vivo knockdown of prom3 in zebrafish alters the number and length of monocilia in the Kupffer's vesicle, resulting in molecular and anatomical defects in the left-right asymmetry. These distinct loss-of-function approaches in two biological systems reveal that prom proteins are critical for the integrity and function of cilia. Our data provide new insights into ciliogenesis and might be of particular interest for investigations of the etiologies of ciliopathies.

scholarly journals Ciliary subcellular localization of TGR5 determines the cholangiocyte functional response to bile acid signaling

2013 ◽  
Vol 304 (11) ◽  
pp. G1013-G1024 ◽  
Author(s):  
Anatoliy I. Masyuk ◽  
Bing Q. Huang ◽  
Brynn N. Radtke ◽  
Gabriella B. Gajdos ◽  
Patrick L. Splinter ◽  
...  
Keyword(s):  
Bile Acid ◽  
Functional Response ◽  
Primary Cilia ◽  
Intracellular Camp ◽  
Ciliated Cells ◽  
Functional Responses ◽  
Polycystic Liver ◽  
A Cell ◽  
And Function ◽  
In Cells

TGR5, the G protein-coupled bile acid receptor that transmits bile acid signaling into a cell functional response via the intracellular cAMP signaling pathway, is expressed in human and rodent cholangiocytes. However, detailed information on the localization and function of cholangiocyte TGR5 is limited. We demonstrated that in human (H69 cells) and rat cholangiocytes, TGR5 is localized to multiple, diverse subcellular compartments, with its strongest expression on the apical plasma, ciliary, and nuclear membranes. To evaluate the relationship between ciliary TGR5 and the cholangiocyte functional response to bile acid signaling, we used a model of ciliated and nonciliated H69 cells and demonstrated that TGR5 agonists induce opposite changes in cAMP and ERK levels in cells with and without primary cilia. The cAMP level was increased in nonciliated cholangiocytes but decreased in ciliated cells. In contrast, ERK signaling was induced in ciliated cholangiocytes but suppressed in cells without cilia. TGR5 agonists inhibited proliferation of ciliated cholangiocytes but activated proliferation of nonciliated cells. The observed differential effects of TGR5 agonists were associated with the coupling of TGR5 to Gαi protein in ciliated cells and Gαs protein in nonciliated cholangiocytes. The functional responses of nonciliated and ciliated cholangiocytes to TGR5-mediated bile acid signaling may have important pathophysiological significance in cilia-related liver disorders (i.e., cholangiociliopathies), such as polycystic liver disease. In summary, TGR5 is expressed on diverse cholangiocyte compartments, including a primary cilium, and its ciliary localization determines the cholangiocyte functional response to bile acid signaling.

scholarly journals Polycystic Diseases in Visceral Organs

2011 ◽  
Vol 2011 ◽  
pp. 1-7 ◽  
Author(s):  
Shakila Abdul-Majeed ◽  
Surya M. Nauli
Keyword(s):  
Mammalian Cells ◽  
Primary Cilia ◽  
Polycystic Ovarian Syndrome ◽  
Cyst Formation ◽  
Apical Surface ◽  
Polycystic Kidney ◽  
Pancreas Disease ◽  
Polycystic Liver ◽  
And Function ◽  
Ovarian Syndrome

Primary cilia are nonmotile, microtubule-based, antenna-like organelles projecting from the apical surface of most mammalian cells. Elegant studies have established the importance of ciliary structure and function in signal transduction and the sensory roles of cilia in maintaining healthy cellular state. In particular, dysfunctional cilia have been implicated in a large number of diseases mainly characterized by the presence of fluid-filled cysts in various organs. Aside from polycystic kidney disease (PKD), however, the roles of cilia in polycystic liver disease (PLD), polycystic pancreas disease (PPD), and polycystic ovarian syndrome (PCOS) are still very vague. In addition, although gender and sex hormones are known to regulate cyst formation, their roles in regulating physiological functions of cilia need to be further explored.

scholarly journals Signaling compartment at the ciliary tip is formed and maintained by intraflagellar transport and functions as sensitive salt detector

2019 ◽  
Author(s):  
Servaas N. van der Burght ◽  
Suzanne Rademakers ◽  
Jacque-Lynne Johnson ◽  
Chunmei Li ◽  
Gert-Jan Kremers ◽  
...  
Keyword(s):  
Primary Cilia ◽  
Physiological Function ◽  
Intraflagellar Transport ◽  
Distal Region ◽  
Central Question ◽  
Membrane Diffusion ◽  
High Concentration ◽  
C Elegans ◽  
Cgmp Signaling ◽  
And Function

AbstractPrimary cilia are ubiquitous antenna-like organelles that mediate cellular signaling and represent hotspots for human diseases termed ciliopathies. How signaling subcompartments are established within the microtubule-based organelle, and for example support Hedgehog or cGMP signal transduction pathways, remains a central question. Here we show that a C. elegans salt-sensing receptor type guanylate cyclase, GCY-22, accumulates at a high concentration within the distal region of the cilium. This receptor uses DAF-25 (Ankmy2 in mammals) to cross the transition zone (TZ) membrane diffusion barrier in the proximal-most region of the ciliary axoneme. Targeting of GCY-22 to the ciliary tip is dynamic, requiring the cargo-mobilizing intraflagellar transport (IFT) system. Disruption of transit across the TZ barrier or IFT trafficking causes GCY-22 protein mislocalization and defects in the formation, maintenance, and function of the ciliary tip compartment required for chemotaxis to low NaCl concentrations. Together, our findings reveal how a previously undescribed cilium tip cGMP signaling compartment is established and contributes to the physiological function of a primary cilium.

scholarly journals Dynein-2 intermediate chains play crucial but distinct roles in primary cilia formation and function

2018 ◽  
Author(s):  
Laura Vuolo ◽  
Nicola L. Stevenson ◽  
Kate J. Heesom ◽  
David J. Stephens
Keyword(s):  
Transition Zone ◽  
Cell Lines ◽  
Primary Cilia ◽  
Point Mutations ◽  
Intraflagellar Transport ◽  
Jeune Syndrome ◽  
Cilia Formation ◽  
Microtubule Motor ◽  
And Function ◽  
Intermediate Chains

AbstractThe dynein-2 microtubule motor is the retrograde motor for intraflagellar transport. Mutations in dynein-2 components cause skeletal ciliopathies, notably Jeune syndrome. Dynein-2 comprises a heterodimer of two non-identical intermediate chains, WDR34 and WDR60. Here, we use knockout cell lines to demonstrate that each intermediate chain has a distinct role in cilia function. Both proteins are required to maintain a functional transition zone and for efficient bidirectional intraflagellar transport, only WDR34 is essential for axoneme extension. In contrast, only WDR60 is essential for co-assembly of the other subunits. Furthermore, WDR60 cannot compensate for loss of WDR34 or vice versa. This work defines a functional asymmetry to match the subunit asymmetry within the dynein-2 motor. Analysis of causative point mutations in WDR34 and WDR60 can partially restore function to knockout cells. Our data show that Jeune syndrome is caused by defects in transition zone architecture as well as intraflagellar transport.SUMMARYHere, Vuolo and colleagues use engineered knockout human cell lines to define roles for dynein-2 intermediate chains. WDR34 is required for axoneme extension, while WDR60 is not. Both subunits are required for cilia transition zone organization and bidirectional intraflagellar transport.

scholarly journals The IFT-A complex regulates Shh signaling through cilia structure and membrane protein trafficking

2012 ◽  
Vol 197 (6) ◽  
pp. 789-800 ◽  
Author(s):  
Karel F. Liem ◽  
Alyson Ashe ◽  
Mu He ◽  
Peter Satir ◽  
Jennifer Moran ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Sonic Hedgehog ◽  
Protein Trafficking ◽  
Primary Cilia ◽  
Intraflagellar Transport ◽  
Loss Of Function ◽  
Shh Signaling ◽  
Shh Pathway ◽  
Membrane Protein Trafficking ◽  
Ectopic Activation

Two intraflagellar transport (IFT) complexes, IFT-A and IFT-B, build and maintain primary cilia and are required for activity of the Sonic hedgehog (Shh) pathway. A weak allele of the IFT-A gene, Ift144, caused subtle defects in cilia structure and ectopic activation of the Shh pathway. In contrast, strong loss of IFT-A, caused by either absence of Ift144 or mutations in two IFT-A genes, blocked normal ciliogenesis and decreased Shh signaling. In strong IFT-A mutants, the Shh pathway proteins Gli2, Sufu, and Kif7 localized correctly to cilia tips, suggesting that these pathway components were trafficked by IFT-B. In contrast, the membrane proteins Arl13b, ACIII, and Smo failed to localize to primary cilia in the absence of IFT-A. We propose that the increased Shh activity seen in partial loss-of-function IFT-A mutants may be a result of decreased ciliary ACIII and that the loss of Shh activity in the absence of IFT-A is a result of severe disruptions of cilia structure and membrane protein trafficking.

scholarly journals STAM and Hrs Down-Regulate Ciliary TRP Receptors

2007 ◽  
Vol 18 (9) ◽  
pp. 3277-3289 ◽  
Author(s):  
Jinghua Hu ◽  
Samuel G. Wittekind ◽  
Maureen M. Barr
Keyword(s):  
Growth Factor ◽  
Mammalian Cells ◽  
Primary Cilia ◽  
Membrane Receptors ◽  
Sensory Transduction ◽  
Kinase Substrate ◽  
C Elegans ◽  
Early Endosomes ◽  
Transient Receptor ◽  
And Function

Cilia are endowed with membrane receptors, channels, and signaling components whose localization and function must be tightly controlled. In primary cilia of mammalian kidney epithelia and sensory cilia of Caenorhabditis elegans neurons, polycystin-1 (PC1) and transient receptor polycystin-2 channel (TRPP2 or PC2), function together as a mechanosensory receptor-channel complex. Despite the importance of the polycystins in sensory transduction, the mechanisms that regulate polycystin activity and localization, or ciliary membrane receptors in general, remain poorly understood. We demonstrate that signal transduction adaptor molecule STAM-1A interacts with C. elegans LOV-1 (PC1), and that STAM functions with hepatocyte growth factor–regulated tyrosine kinase substrate (Hrs) on early endosomes to direct the LOV-1-PKD-2 complex for lysosomal degradation. In a stam-1 mutant, both LOV-1 and PKD-2 improperly accumulate at the ciliary base. Conversely, overexpression of STAM or Hrs promotes the removal of PKD-2 from cilia, culminating in sensory behavioral defects. These data reveal that the STAM-Hrs complex, which down-regulates ligand-activated growth factor receptors from the cell surface of yeast and mammalian cells, also regulates the localization and signaling of a ciliary PC1 receptor-TRPP2 complex.

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