The discovery and exploration of a universal targeting mechanism in eukaryotic cells

A wide range of eukaryotic organisms generate motile cilia and flagella. These slender organelles beat rhythmically to move the surrounding fluid or to propel cells in aqueous environment. Organisms use these powerful yet nimble organelles to forage, evade, adapt and mate. The machinery that drives this tightly controlled movement is the sophisticated microtubule-based axoneme. As it is critical for the survival of individual species, this machinery has largely been preserved to the molecular level throughout evolution. Proteomic studies have shown that most proteins in this biological machine consist of molecular modules commonly used in the cell body. But the usage of these modules is clearly diverged in many cases. This defined machinery with diverged applications provides an opportunity to understand the true capacity of the conserved modules. One example is the radial spoke (RS) that controls the oscillatory beating. This macromolecular complex contains complementary molecular modules that are responsible for localizing cAMP-dependent protein kinase (PKA) in the cell body. However, the RS does not have the features that account for the effector mechanisms of PKA and thus the mechanism discovered for localizing PKA has a broader role that has previously not been recognized. The work described in this dissertation discovered that the core of the RS utilizes two similar sets of PKA anchoring modules for four distinct effector mechanisms that underlie the assembly and function of this regulatory complex. These results elucidate the function of this complex and are applicable to more than 600 diverged proteins that also share the docking module of PKA. Some of them have been shown to play vital roles in myriads of cellular reactions ranging from flagellar beating to trans-Golgi trafficking to chromosome modifications. Founded on this discovery, new reagents and assays were engineered. These tools could be used for the exploration of proteins with similar docking and anchoring modules. Together, these findings will accelerate the advancements in the field of anchoring and docking of proteins in the cell

\u3cem\u3eChlamydomonas\u3c/em\u3e mutants display reversible deficiencies in flagellar beating and axonemal assembly

Axonemal complexes in flagella are largely prepackaged in the cell body. As such, one mutation often results in the absence of the co-assembled components and permanent motility deficiencies. For example, a Chlamydomonas mutant defective in RSP4 in the radial spoke (RS), which is critical for bend propagation, has paralyzed flagella that also lack the paralogue RSP6 and three additional RS proteins. Intriguingly, recent studies showed that several mutant strains contain a mixed population of swimmers and paralyzed cells despite their identical genetic background. Here we report a cause underlying these variations. Two new mutants lacking RSP6 swim processively and other components appear normally assembled in early log phase indicating that, unlike RSP4, this paralogue is dispensable. However, swimmers cannot maintain the typical helical trajectory and reactivated cell models tend to spin. Interestingly the motile fraction and the spokehead content dwindle during stationary phase. These results suggest that (1) intact RS is critical for maintaining the rhythm of oscillatory beating and thus the helical trajectory; (2) assembly of the axonemal complex with subtle defects is less efficient and the inefficiency is accentuated in compromised conditions, leading to reversible dyskinesia. Consistently, several organisms only possess one RSP4/6 gene. Gene duplication in Chlamydomonas enhances RS assembly to maintain optimal motility in various environments

Myc-binding Protein Orthologue Interacts with AKAP240 In the Central Pair Apparatus of the \u3cem\u3eChlamydomonas\u3c/em\u3e Flagella

Background Flagella and cilia are fine thread-like organelles protruding from cells that harbour them. The typical ‘9 + 2’ cilia confer motility on these cells. Although the mechanistic details of motility remain elusive, the dynein-driven motility is regulated by various kinases and phosphatases. A-kinase anchoring proteins (AKAPs) are scaffolds that bind to a variety of such proteins. Usually, they are known to possess a dedicated domain that in vitro interacts with the regulatory subunits (RI and RII) present in the cAMP-dependent protein kinase (PKA) holoenzyme. These subunits conventionally harbour contiguous stretches of a.a. residues that reveal the presence of the Dimerization Docking (D/D) domain, Catalytic interface domain and cAMP-Binding domain. The Chlamydomonas reinhardtii flagella harbour two AKAPs; viz., the radial spoke AKAP97 or RSP3 and the central pair AKAP240. Both these were identified on the basis of their RII-binding property. Interestingly, AKAP97 binds in vivo to two RII-like proteins (RSP7 and RSP11) that contain only the D/D domain. Results We found a Chlamydomonas Flagellar Associated Protein (FAP174) orthologous to MYCBP-1, a protein that binds to organellar AKAPs and Myc onco-protein. An in silico analysis shows that the N-terminus of FAP174 is similar to those RII domain-containing proteins that have binding affinities to AKAPs. Binding of FAP174 was tested with the AKAP97/RSP3 using in vitro pull down assays; however, this binding was rather poor with AKAP97/RSP3. Antibodies were generated against FAP174 and the cellular localization was studied using Western blotting and immunoflourescence in wild type and various flagella mutants. We show that FAP174 localises to the central pair of the axoneme. Using overlay assays we show that FAP174 binds AKAP240 previously identified in the C2 portion of the central pair apparatus. Conclusion It appears that the flagella of Chlamydomonas reinhardtii contain proteins that bind to AKAPs and except for the D/D domain, lack the conventional a.a. stretches of PKA regulatory subunits (RSP7 and RSP11). We add FAP174 to this growing list

The Versatile Molecular Complex Component LC8 Promotes Several Distinct Steps of Flagellar Assembly

LC8 is present in various molecular complexes. However, its role in these complexes remains unclear. We discovered that although LC8 is a subunit of the radial spoke (RS) complex in Chlamydomonas flagella, it was undetectable in the RS precursor that is converted into the mature RS at the tip of elongating axonemes. Interestingly, LC8 dimers bound in tandem to the N-terminal region of a spoke phosphoprotein, RS protein 3 (RSP3), that docks RSs to axonemes. LC8 enhanced the binding of RSP3 N-terminal fragments to purified axonemes. Likewise, the N-terminal fragments extracted from axonemes contained LC8 and putative spoke-docking proteins. Lastly, perturbations of RSP3’s LC8-binding sites resulted in asynchronous flagella with hypophosphorylated RSP3 and defective associations between LC8, RSs, and axonemes. We propose that at the tip of flagella, an array of LC8 dimers binds to RSP3 in RS precursors, triggering phosphorylation, stalk base formation, and axoneme targeting. These multiple effects shed new light on fundamental questions about LC8-containing complexes and axoneme assembly

A Flagellar A-Kinase Anchoring Protein with Two Amphipathic Helices Forms a Structural Scaffold in the Radial Spoke Complex

A-kinase anchoring proteins (AKAPs) contain an amphipathic helix (AH) that binds the dimerization and docking (D/D) domain, RIIa, in cAMP-dependent protein kinase A (PKA). Many AKAPs were discovered solely based on the AH–RIIa interaction in vitro. An RIIa or a similar Dpy-30 domain is also present in numerous diverged molecules that are implicated in critical processes as diverse as flagellar beating, membrane trafficking, histone methylation, and stem cell differentiation, yet these molecules remain poorly characterized. Here we demonstrate that an AKAP, RSP3, forms a dimeric structural scaffold in the flagellar radial spoke complex, anchoring through two distinct AHs, the RIIa and Dpy-30 domains, in four non-PKA spoke proteins involved in the assembly and modulation of the complex. Interestingly, one AH can bind both RIIa and Dpy-30 domains in vitro. Thus, AHs and D/D domains constitute a versatile yet potentially promiscuous system for localizing various effector mechanisms. These results greatly expand the current concept about anchoring mechanisms and AKAPs

A Novel Boosting Technique for Induction Heating Application with ZVS Soft Switching

This paper presents a direct ac-ac converter boosting technique in induction heating (IH) application using half bridge configuration. The main design features are efficiency and user performance. Efficiency is one of the important parameter when designing a converter for induction heating application. The benefits of this direct ac-ac resonant converter under half bridge configuration are reduced number of components, low cost, reduced size of the converter for same power rating and direct conversion solution. Its operating performance and high frequency power regulation characteristics are explained on the basis of simulation. The simulations were obtained by MATLAB/SIMULINK

Endodontic Management of a Severely Dilacerated Mandibular Third Molar: Case Report and Clinical Considerations

This article aims at providing an insight to the clinical modifications required for the endodontic management of severely dilacerated mandibular third molar. A 35-year-old patient was referred for the root canal treatment of the mandibular left third molar. An intraoral periapical radiograph revealed a severe curvature in both the canals. A wide trapezoidal access was prepared following the use of intermediate-sized files for apical preparation. Owing to increased flexibility, Hero Shaper NITI files were used for the biomechanical preparation and single cone obturation was carried out. Third molars owing to their most posterior location-limited access coupled with a severe curvature pose utmost clinical challenges require meticulous skill, advanced technology, and patience to achieve success

Migration of breast cancer cell lines in response to pulmonary laminin 332

Because tumor cell motility is a requirement for metastasis, we hypothesized that lung tissue harbors substances that induce tumor cell migration. MCF-7 breast carcinoma cells exposed to small airway epithelial cells and conditioned medium exhibited dose-dependent tumor cell migration. Among the extracellular matrix proteins in the conditioned medium identified by mass spectrometry, laminin 332 (LM332) had the greatest contribution to the migration of MCF-7 cells. Immunoblotting and immunohistochemistry for LM332-specific chains identified LM332 in the lung and in pulmonary epithelial cells. Antibodies to either LM332 or its integrin receptor inhibited MCF-7 motility, and knockdown of LM332 chains also reduced its migration-inducing activity. Taken together, these findings implicate LM332 as a component of lung tissue that can induce motility in breast carcinoma cells that have been transported to lung during metastasis. Earlier studies on LM332 in tumor progression have examined LM332 expression in tumor cells. This investigation, in comparison, provides evidence that the tumor promoting potential of LM332 may originate in the lung microenvironment rather than in tumor cells alone. Furthermore, this study provides evidence that the motility-inducing properties of the microenvironment can reside in epithelial cells. The findings raise the possibility that LM332 plays a role in the pulmonary metastases of breast carcinoma and may provide a target for antimetastasis therapy

Additional file 1: Figure S1. of Myc-binding protein orthologue interacts with AKAP240 in the central pair apparatus of the Chlamydomonas flagella

Testing the interaction of FAP174 with GST-tagged RSP3 in vitro. (A) Co-expression of FAP174 with GST or GST-tagged RSP3. The first, second and fourth lanes are the crude lysates with independently expressing FAP174, GST and GST-RSP3 proteins (see arrowheads); the third lane is the clone over-expressing both the FAP174 and GST proteins; while, the last lane is the lysate from the clone over-expressing both FAP174 and GST-RSP3 (see arrowheads). (B) FAP174 (arrow at the bottom of the gel) was co-purified with GST-RSP3 (arrow at the top of the gel) but not with GST alone. FT, W and E are Flow-through, Wash and Elutes, respectively. Note that there no interaction between full-length RSP3 and FAP174. (PNG 379 kb

Additional file 1: Figure S1. of Myc-binding protein orthologue interacts with AKAP240 in the central pair apparatus of the Chlamydomonas flagella

Testing the interaction of FAP174 with GST-tagged RSP3 in vitro. (A) Co-expression of FAP174 with GST or GST-tagged RSP3. The first, second and fourth lanes are the crude lysates with independently expressing FAP174, GST and GST-RSP3 proteins (see arrowheads); the third lane is the clone over-expressing both the FAP174 and GST proteins; while, the last lane is the lysate from the clone over-expressing both FAP174 and GST-RSP3 (see arrowheads). (B) FAP174 (arrow at the bottom of the gel) was co-purified with GST-RSP3 (arrow at the top of the gel) but not with GST alone. FT, W and E are Flow-through, Wash and Elutes, respectively. Note that there no interaction between full-length RSP3 and FAP174. (PNG 379 kb