Cytoplasmic Dynein Intermediate Chain Research Articles

Expression and functional analysis of cytoplasmic dynein during spermatogenesis in Portunus trituberculatus

The mechanism of acrosome formation in the crab sperm is a hot topic in crustacean reproduction research. Dynein is a motor protein that performs microtubule-dependent retrograde transport and plays an essential role in spermatogenesis. However, whether cytoplasmic dynein participates in acrosome formation in the crab sperm remains poorly understood. In this study, we cloned the cytoplasmic dynein intermediate chain gene (Pt-DIC) from Portunus trituberculatus testis. Pt-DIC is composed of a p150glued-binding domain, a dynein light chain (DLC)-binding domain, and a dynein heavy chain (DHC)-binding domain. The Pt-DIC gene is widely expressed in different tissues, showing the highest expression in the testis, and it is expressed in different stages of spermatid development, indicating important functions in spermatogenesis. We further observed the colocalization of Pt-DIC and Pt-DHC, Pt-DHC and tubulin, and Pt-DHC and GM130, and the results indicated that cytoplasmic dynein may participate in nuclear shaping and acrosome formation via vesicle transport. In addition, we examined the colocalization of Pt-DHC and a mitochondrion (MT) tracker and that of Pt-DHC and prohibitin (PHB). The results indicated that cytoplasmic dynein participated in mitochondrial transport and mitochondrial degradation. Taken together, these results support the hypothesis that cytoplasmic dynein participates in acrosome formation, nuclear shaping, and mitochondrial transport during spermiogenesis in P. trituberculatus. This study will provide valuable guidance for the artificial fertilization and reproduction of P. trituberculatus.

Downhill Running Decreases the Acetylation of Tubulins and Impairs Autophagosome Degradation in Rat Skeletal Muscle.

This study was designed to probe the effect of downhill running on microtubule acetylation and autophagic flux in rat skeletal muscle. Sprague-Dawley rats were subjected to an exercise protocol of a 90-min downhill run with a slope of -16° and a speed of 16 m·min-1, and then the soleus was sampled at 0, 12, 24, 48, and 72 h after exercise. Protein expression levels of microtubule-associated protein 1 light chain 3 (LC3), p62/sequestosome 1 (p62), α-tubulin, and acetylated α-tubulin (AcK40 α-tubulin) were detected by Western blotting. Alpha-tubulin was costained with AcK40 α-tubulin or cytoplasmic dynein intermediate chain in a single muscle fiber, and LC3 was costained with lysosomal-associated membrane protein 1 in cryosections. To assess autophagic flux in vivo, colchicine or vehicle was injected intraperitoneally 3 d before the exercise experiment, and the protein levels of LC3 and p62 were measured by Western blotting. Downhill running induced a significant increase in the protein levels of LC3-II and p62, whereas the level and proportion of AcK40 α-tubulin were markedly decreased. Furthermore, the amount of dynein on α-tubulin was decreased after downhill running, and autophagosomes accumulated in the middle of myofibrils. Importantly, LC3-II flux was decreased after downhill running compared with that in the control group. A bout of downhill running decreases microtubule acetylation, which may impair dynein recruitment and autophagosome transportation, causing blocked autophagic flux.

Loss of dynein-2 intermediate chain Wdr34 results in defects in retrograde ciliary protein trafficking and Hedgehog signaling in the mouse.

The Wdr34 gene encodes an intermediate chain of cytoplasmic dynein 2, the motor for retrograde intraflagellar transport (IFT) in primary cilia. Although mutations in human WDR34 have recently been reported, the association of WDR34 function with Hedgehog (Hh) signaling has not been established, and actual cilia defects in the WDR34 mutant cells have also not been completely characterized. In the present study, we show that Wdr34 mutant mice die in midgestation and exhibit open brain and polydactyly phenotypes. Several Hh-dependent ventral neural cell types are not specified in the mutant neural tube. The expression of the direct Hh targets, Gli1 and Patched 1, is inhibited, while the expression of limb patterning genes that are normally inhibited by the Gli3 repressor is anteriorly expanded in mutant limbs. Comparison of cilia phenotype and function among wild type, Dnchc2 (dynein 2 heavy chain), and Wdr34 mutant cells demonstrates that cilia in both Dnchc2 and Wdr34 mutant cells are stumpy. Several ciliary proteins examined abnormally accumulate in the cilia of both mutant cells. Consistent with its function, overexpressed Wdr34 is occasionally localized to cilia, and Wdr34 is required for the ciliary localization of dynein 2 light intermediate chain Lic3. More interestingly, we show that both Dnchc2 and Wdr34 act between Smo and Gli2/Gli3 in the Hh pathway. Therefore, like Dnchc2, Wdr34 is required for ciliogenesis, retrograde ciliary protein trafficking, and the regulation of Gli2/Gli3 activators and repressors. Furthermore, both Wdr34 and Dnchc2 promote microtubule growth, a novel dynein 2 function in a non-cilia structure.

PDK1–Akt pathway regulates radial neuronal migration and microtubules in the developing mouse neocortex

Neurons migrate a long radial distance by a process known as locomotion in the developing mammalian neocortex. During locomotion, immature neurons undergo saltatory movement along radial glia fibers. The molecular mechanisms that regulate the speed of locomotion are largely unknown. We now show that the serine/threonine kinase Akt and its activator phosphoinositide-dependent protein kinase 1 (PDK1) regulate the speed of locomotion of mouse neocortical neurons through the cortical plate. Inactivation of the PDK1-Akt pathway impaired the coordinated movement of the nucleus and centrosome, a microtubule-dependent process, during neuronal migration. Moreover, the PDK1-Akt pathway was found to control microtubules, likely by regulating the binding of accessory proteins including the dynactin subunit p150(glued) Consistent with this notion, we found that PDK1 regulates the expression of cytoplasmic dynein intermediate chain and light intermediate chain at a posttranscriptional level in the developing neocortex. Our results thus reveal an essential role for the PDK1-Akt pathway in the regulation of a key step of neuronal migration.

Identification of the T-complex protein as a binding partner for newly synthesized cytoplasmic dynein intermediate chain 2

Cytoplasmic dynein is a macromolecular motor complex with diverse functions in eukaryotic cells. Dynein plays essential roles in intracellular transport of organelles and mitosis, mediated in part by interactions between the dynein intermediate chain 2 (IC-2) subunits and adapter proteins that bind specific cargos. In experiments to identify phosphorylation-dependent binding partners for IC-2 we instead identified a phosphorylation-independent binding partner, the cytosolic chaperonin containing T complex protein 1 (CCT). CCT consists of eight subunits (CCT1-8) and facilitates folding of a subset of newly synthesized proteins. We confirmed interactions between IC-2 and CCT5 and CCT8 in co-immunoprecipitation experiments and determined that the C-terminal half of IC-2 is necessary and sufficient to bind CCT8. Interestingly, co-immunoiprecipitation of IC-2 and CCT is abolished by prior cycloheximide treatment of cells, suggesting that CCT participates in folding of nascent IC-2. In vitro translation experiments employing recombinant CCT complex demonstrated that CCT is able to bind newly synthesized IC-2 after release from the ribosome consistent with a role in folding of IC-2.

Analysis of dynein intermediate chains, light intermediate chains and light chains in a cohort of hereditary peripheral neuropathies.

The cytoplasmic dynein heavy chain (DYNC1H1) gene has been increasingly associated with neurodegenerative disorders including axonal Charcot-Marie-Tooth disease (CMT2), intellectual disability and malformations of cortical development. In addition, evidence from mouse models (Loa, catabolite repressor-activator (Cra) and Sprawling (Swl)) has shown that mutations in Dync1h1 cause a range of neurodegenerative phenotypes with motor and sensory neuron involvement. In this current study, we examined the possible contribution of other cytoplasmic dynein subunits that bind to DYNC1H1 as a cause of inherited peripheral neuropathy. We focused on screening the cytoplasmic dynein intermediate, light intermediate and light chain genes in a cohort of families with inherited peripheral neuropathies. Nine genes were screened and ten variants were detected, but none was identified as pathogenic, indicating that cytoplasmic dynein intermediate, light intermediate and light chains are not a cause of neuropathy in our cohort.

Cloning and characterization of cytoplasmic dynein intermediate chain in Fenneropenaeus chinensis and its essential role in white spot syndrome virus infection

To investigate the role of cytoplasmic dynein in white spot syndrome virus (WSSV) infection, the full-length cDNA of cytoplasmic dynein intermediate chain (FcDYNCI) was cloned in Fenneropenaeus chinensis, which consists of 2582 bp and encodes a polypeptide of 660 amino acids. Sequence analysis and multiple sequence alignment displayed that FcDYNCI was a member of cytoplasmic dynein 1 family. The FcDYNCI mRNA was most highly expressed in hemocytes, which was significantly up-regulated post WSSV infection. At 12 h post infection (hpi), confocal microscopic observation showed that WSSV could be co-localized with cytoplasmic dynein in hemocytes. After silencing by specific FcDYNCI dsRNA, the FcDYNCI mRNA level and the protein amount of FcDYNCI in hemocytes both exhibited a significant reduction, and the expression levels of three WSSV genes ie1, wsv477 and vp28 all exhibited the greatest decreases at 24 hpi. These results suggested that cytoplasmic dynein was involved in WSSV infection.

Epidermal Growth Factor Stimulates Extracellular-Signal Regulated Kinase Phosphorylation of a Novel Site on Cytoplasmic Dynein Intermediate Chain 2

Extracellular-signal regulated kinase (ERK) signaling is required for a multitude of physiological and patho-physiological processes. However, the identities of the proteins that ERK phosphorylates to elicit these responses are incompletely known. Using an affinity purification methodology of general utility, here we identify cytoplasmic dynein intermediate chain 2 (DYNC1I-2, IC-2) as a novel substrate for ERK following epidermal growth factor receptor stimulation of fibroblasts. IC-2 is a subunit of cytoplasmic dynein, a minus-end directed motor protein necessary for transport of diverse cargos along microtubules. Emerging data support the hypothesis that post-translational modification regulates dynein but the signaling mechanisms used are currently unknown. We find that ERK phosphorylates IC-2 on a novel, highly conserved Serine residue proximal to the binding site for the p150Glued subunit of the cargo adapter dynactin. Surprisingly, neither constitutive phosphorylation nor a phosphomimetic substitution of this Serine influences binding of p150Glued to IC-2. These data suggest that ERK phosphorylation of IC-2 regulates dynein function through mechanisms other than its interaction with dynactin.

The Usher syndrome 1G protein SANS participates in the transport of ciliary cargo in photoreceptor cells

Human Usher syndrome (USH) is the most common form of combined deaf-blindness, characterized by profound congenital deafness, constant vestibular dysfunction and pre-pubertal onset of retinitis pigmentosa. The USH1G protein SANS (scaffold protein containing ankyrin repeats and SAM domain) is associated with microtubules and mediates a transport related periciliary protein network in photoreceptor cells. Here we aim to enlighten the involvement of SANS in ciliary transport of photoreceptor cells by identifying proteins associated with SANS in transport complexes. In Y2H screen of retinal cDNA library we identified the direct binding of SANS to dynactin-1 (p150Glued), a subunit of the dynactin complex and cargo linker to the cytoplasmic dynein motor. This interaction was validated in complementary interaction assays in vitro and in cell culture. In addition, we demonstrated the integration of SANS into the cytoplasmic dynein transport complex by GST-pull down of SANS with cytoplasmic dynein intermediate chain (cDIC74). Correlative immunofluorescence and -electron microscopy analyses of photoreceptor inner segments revealed co-localization of essential dynein-dynactin components and SANS at microtubules, the known transport routes for opsin bearing vesicles. Co-immunoprecipitation revealed that SANS is part of an opsin containing protein complex together with transport regulating rab-GTPases. Finally, the analysis of SANS deficient mice revealed altered distribution of opsin in the inner segment, indicating a transport delay. In conclusion we identified SANS as a component of the cytoplasmic dynein motor complex which is crucial for opsin transport to photoreceptor connecting cilia. Defects in these transport mechanisms lead to retinal degeneration as characteristic for USH1G patients.

Drosophila Dynein Intermediate Chain Gene, Dic61B, Is Required for Spermatogenesis

This study reports the identification and characterization of a novel gene, Dic61B, required for male fertility in Drosophila. Complementation mapping of a novel male sterile mutation, ms21, isolated in our lab revealed it to be allelic to CG7051 at 61B1 cytogenetic region, since two piggyBac insertion alleles, CG7051c05439 and CG7051f07138 failed to complement. CG7051 putatively encodes a Dynein intermediate chain. All three mutants, ms21, CG7051c05439 and CG7051f07138, exhibited absolute recessive male sterility with abnormally coiled sperm axonemes causing faulty sperm individualization as revealed by Phalloidin staining in Don Juan-GFP background. Sequencing of PCR amplicons uncovered two point mutations in ms21 allele and confirmed the piggyBac insertions in CG7051c05439 and CG7051f07138 alleles to be in 5′UTR and 4th exon of CG7051 respectively, excision of which reverted the male sterility. In situ hybridization to polytene chromosomes demonstrated CG7051 to be a single copy gene. RT-PCR of testis RNA revealed defective splicing of the CG7051 transcripts in mutants. Interestingly, expression of cytoplasmic dynein intermediate chain, α, β, γ tubulins and α-spectrin was normal in mutants while ultra structural studies revealed defects in the assembly of sperm axonemes. Bioinformatics further highlighted the homology of CG7051 to axonemal dynein intermediate chain of various organisms, including DNAI1 of humans, mutations in which lead to male sterility due to immotile sperms. Based on these observations we conclude that CG7051 encodes a novel axonemal dynein intermediate chain essential for male fertility in Drosophila and rename it as Dic61B. This is the first axonemal Dic gene of Drosophila to be characterized at molecular level and shown to be required for spermatogenesis.

Tandem reporter assay for myristoylated proteins post‐translationally (TRAMPP) identifies novel substrates for post‐translational myristoylation: PKC∊, a case study

Myristoylation, the addition of a 14-carbon fatty acid to the N-terminal glycine of a protein, is key to protein-membrane and protein-protein interactions. Typically, myristoylation occurs cotranslationally; however, post-translational myristoylation of caspase-cleaved proteins is now emerging as a well-established protein modification and as a novel regulator of apoptosis. To identify additional post-translationally myristoylated proteins, we engineered a plasmid vector encoding for a caspase-cleavable reporter protein named tandem reporter assay for myristoylation of proteins post-translationally (TRAMPP). pTRAMPP consists of tdTomato-DEVD-"test myristoylation sequence"-enhanced green fluorescent protein (EGFP). After induction of apoptosis, the reporter protein is cleaved by caspases, which frees a new N-terminal glycine residue attached to EGFP that can be myristoylated. We used pTRAMPP in appropriately transfected cells to identify 7 post-translationally myristoylated proteins. First, we confirmed the post-translational myristoylation of two previously identified putative substrates, cytoplasmic dynein intermediate chain 2A and PKCε (ctPKCε), and identified 5 more caspase-cleaved potential substrates for myristoylation that include the antiapoptotic regulator of apoptosis, Mcl-1, and the causative agent of Huntington's disease, huntingtin protein. Further investigation revealed that post-translationally myristoylated ctPKCε localized to membranes and increased Erk signaling and degradation of the proapoptotic protein Bim, which prevented a significant loss of mitochondrial potential of 17% over nonmyristoylated ctPKCε in HeLa cells in the presence of apoptotic stimuli. Taken together, these findings suggest a possible antiapoptotic role for post-translationally myristoylated caspase-cleaved ctPKCε.

Differential Proteomic Analysis of Platelets Suggested Possible Signal Cascades Network in Platelets Treated with Salvianolic Acid B

BackgroundSalvianolic acid B (SB) is an active component isolated from Danshen, a traditional Chinese medicine widely used for the treatment of cardiovascular disorders. Previous study suggested that SB might inhibit adhesion as well as aggregation of platelets by a mechanism involving the integrin α2β1. But, the signal cascades in platelets after SB binding are still not clear.Methodology/Principal FindingsIn the present study, a differential proteomic analysis (two-dimensional electrophoresis) was conducted to check the protein expression profiles of rat platelets with or without treatment of SB. Proteins altered in level after SB exposure were identified by MALDI-TOF MS/MS. Treatment of SB caused regulation of 20 proteins such as heat shock-related 70 kDa protein 2 (hsp70), LIM domain protein CLP-36, copine I, peroxiredoxin-2, coronin-1 B and cytoplasmic dynein intermediate chain 2C. The regulation of SB on protein levels was confirmed by Western blotting. The signal cascades network induced by SB after its binding with integrin α2β1 was predicted. To certify the predicted network, binding affinity of SB to integrin α2β1 was checked in vitro and ex vivo in platelets. Furthermore, the effects of SB on protein levels of hsp70, coronin-1B and intracellular levels of Ca(2+) and reactive oxygen species (ROS) were checked with or without pre-treatment of platelets using antibody against integrin α2β1. Electron microscopy study confirmed that SB affected cytoskeleton structure of platelets.Conclusions/SignificanceIntegrin α2β1 might be one of the direct target proteins of SB in platelets. The signal cascades network of SB after binding with integrin α2β1 might include regulation of intracellular Ca(2+) level, cytoskeleton-related proteins such as coronin-1B and cytoskeleton structure of platelets.

Neurodegenerative Mutation in Cytoplasmic Dynein Alters Its Organization and Dynein-Dynactin and Dynein-Kinesin Interactions

A single amino acid change, F580Y (Legs at odd angles (Loa), Dync1h1Loa), in the highly conserved and overlapping homodimerization, intermediate chain, and light intermediate chain binding domain of the cytoplasmic dynein heavy chain can cause severe motor and sensory neuron loss in mice. The mechanism by which the Loa mutation impairs the neuron-specific functions of dynein is not understood. To elucidate the underlying molecular mechanisms of neurodegeneration arising from this mutation, we applied a cohort of biochemical methods combined with in vivo assays to systemically study the effects of the mutation on the assembly of dynein and its interaction with dynactin. We found that the Loa mutation in the heavy chain leads to increased affinity of this subunit of cytoplasmic dynein to light intermediate and a population of intermediate chains and a suppressed association of dynactin to dynein. These data suggest that the Loa mutation drives the assembly of cytoplasmic dynein toward a complex with lower affinity to dynactin and thus impairing transport of cargos that tether to the complex via dynactin. In addition, we detected up-regulation of kinesin light chain 1 (KLC1) and its increased association with dynein but reduced microtubule-associated KLC1 in the Loa samples. We provide a model describing how up-regulation of KLC1 and its interaction with cytoplasmic dynein in Loa could play a regulatory role in restoring the retrograde and anterograde transport in the Loa neurons.

Correction: Mouse Cytoplasmic Dynein Intermediate Chains: Identification of New Isoforms, Alternative Splicing and Tissue Distribution of Transcripts

Correction: Mouse Cytoplasmic Dynein Intermediate Chains: Identification of New Isoforms, Alternative Splicing and Tissue Distribution of Transcripts

Mouse Cytoplasmic Dynein Intermediate Chains: Identification of New Isoforms, Alternative Splicing and Tissue Distribution of Transcripts

BackgroundIntracellular transport of cargoes including organelles, vesicles, signalling molecules, protein complexes, and RNAs, is essential for normal function of eukaryotic cells. The cytoplasmic dynein complex is an important motor that moves cargos along microtubule tracks within the cell. In mammals this multiprotein complex includes dynein intermediate chains 1 and 2 which are encoded by two genes, Dync1i1 and Dync1i2. These proteins are involved in dynein cargo binding and dynein complexes with different intermediate chains bind to specific cargoes, although the mechanisms to achieve this are not known. The DYNC1I1 and DYNC1I2 proteins are translated from different splice isoforms, and specific forms of each protein are essential for the function of different dynein complexes in neurons.Methodology/Principal FindingsHere we have undertaken a systematic survey of the dynein intermediate chain splice isoforms in mouse, basing our study on mRNA expression patterns in a range of tissues, and on bioinformatics analysis of mouse, rat and human genomic and cDNA sequences. We found a complex pattern of alternative splicing of both dynein intermediate chain genes, with maximum complexity in the embryonic and adult nervous system. We have found novel transcripts, including some with orthologues in human and rat, and a new promoter and alternative non-coding exon 1 for Dync1i2.Conclusions/SignificanceThese data, including the cloned isoforms will be essential for understanding the role of intermediate chains in the cytoplasmic dynein complex, particularly their role in cargo binding within individual tissues including different brain regions.

Identification of DH IC-2 as a HIF-1 independent protein involved in the adaptive response to hypoxia in tumor cells: A putative role in metastasis

The master regulator of the adaptive response to hypoxia is HIF-1. However, while some data show that HIF-1 can control more than 80% of the genes induced under hypoxia, other experiments clearly demonstrate that a part of the hypoxic response is independent of HIF-1. The goal of this study was to identify some of these HIF-1 independent factors and to investigate their functional role in the adaptation of tumor cells to hypoxia. We show that the cytoplasmic dynein intermediate chain 2 (DH IC-2), a component of an intracellular ATPase minus-end directed tubulin-based motile complex, was stabilized and post-translationally modified under hypoxia in a HIF-1 independent way. We identified this modification as a phosphorylation by protein kinase C, which is inhibited under hypoxia. In parallel, the migration of HepG2 cells was enhanced under hypoxia. Cell migration was also increased, to the same extent, by the invalidation of DH IC-2 using siRNA. Taken together, these results suggest that under hypoxia, a specific modification of DH IC-2 may modulate its activity, and in turn promote cell migration. These results are important to better understand cancer development since they highlight a HIF-1 independent mechanism, which may be involved in metastasis.

Intermediate chain subunit as a probe for cytoplasmic dynein function: Biochemical analyses and live cell imaging in PC12 cells

Cytoplasmic dynein 1 is a multi-subunit motor protein responsible for microtubule minus end-directed transport in axons. The cytoplasmic dynein intermediate chain subunit has a scaffold-like role in the dynein complex; it directly binds to four of the other five subunits, the heavy chain and the three light chains. The intermediate chain also binds the p150 subunit of dynactin, a protein that is essential for many dynein functions. We reexamined the generation of rat cytoplasmic dynein intermediate chain isoforms by the alternative splicing of the two genes that encode this subunit and identified an additional splicing site in intermediate chain gene 1. We reinvestigated the expression of the intermediate chain 1 isoforms in cultured cells and tissues. The Loa mouse, which is homozygote lethal, contains a missense mutation in the region of the cytoplasmic dynein heavy chain gene that binds the intermediate chain. Protein binding studies showed that all six intermediate chains were able to bind to the mutated heavy chain. GFP-tagged intermediate chains were constructed and PC12 cell lines with stable expression of the fusion proteins were established. Live cell imaging and comparative immunocytochemical analyses show that dynein is enriched in the actin rich region of growth cones.

Identification of a Novel Region of the Cytoplasmic Dynein Intermediate Chain Important for Dimerization in the Absence of the Light Chains

Cytoplasmic dynein is the multisubunit protein complex responsible for many microtubule-based intracellular movements. Its cargo binding domain consists of dimers of five subunits: the intermediate chains, the light intermediate chains, and the Tctex1, Roadblock, and LC8 light chains. The intermediate chains have a key role in the dynein complex. They bind the three light chains and the heavy chains, which contain the motor domains, but little is known about how the two intermediate chains interact. There are six intermediate chain isoforms, and it has been hypothesized that different isoforms may regulate specific dynein functions. However, there are little data on the potential combinations of the intermediate chain isoforms in the dynein complexes. We used co-immunoprecipitation analyses to demonstrate that all combinations of homo- and heterodimers of the six intermediate chains are possible. Therefore the formation of dynein complexes with different combinations of isoforms is not limited by interaction between the various intermediate chains. We further sought to identify the domain necessary for the dimerization of the intermediate chains. Analysis of a series of truncation and deletion mutants showed that a 61-amino-acid region is necessary for dimerization of the intermediate chain. This region does not include the N-terminal coiled-coil, the C-terminal WD repeat domain, or the three different binding sites for the Tctex1, LC8, and Roadblock light chains. Analytical gel filtration and covalent cross-linking of purified recombinant polypeptides further demonstrated that the intermediate chains can dimerize in vitro in the absence of the light chains.

Identification of Dynamic Proteome Changes Upon Ligand Activation of Trk-Receptors Using Two-dimensional Fluorescence Difference Gel Electrophoresis and Mass Spectrometry

The TrkA and TrkB tyrosine kinases are members of the neurotrophin receptor family and mediate survival, differentiation, growth, and apoptosis of neurons in response to stimulation by their ligands, NGF and BDNF, respectively. Expression levels of TrkA/TrkB are important prognostic factors in a variety of embryonal tumors including neuroblastoma, the most common solid tumor of childhood. Because TrkA/TrkB exhibit a high level of sequence similarity and use overlapping pathways for signal transduction, the existence of specific effector molecules crucial for receptor and cell-type-specific response is likely. To identify these effectors by analyzing biological effects of TrkA and TrkB activation in a defined model, we performed a proteome study using the human neuroblastoma SY5Y cell line stably transfected with the TrkA or TrkB cDNA. The use of the recently introduced DIGE (fluorescence two-dimensional difference gel electrophoresis) system (Amersham Biosciences, Piscataway, NJ) allowed us to monitor differences in protein expression between samples in one gel. Proteomic changes were monitored in a time course of 0, 0.5, 1, 6, and 24 h following receptor activation. Using MALDI mass spectrometry, we identified, respectively, 22 and 9 differentially expressed proteins upon the addition of neurotrophin in SY5Y-TrkB and SY5Y-TrkA cells. Functional assignment revealed that the majority of these proteins are involved in organization and maintenance of cellular structures.

Solution Structure of the Tctex1 Dimer Reveals a Mechanism for Dynein-Cargo Interactions

Tctex1 is a light chain found in both cytoplasmic and flagellar dyneins and is involved in many fundamental cellular activities, including rhodopsin transport within photoreceptors, and may function in the non-Mendelian transmission of t haplotypes in mice. Here, we present the NMR solution structure for the Tctex1 dimer from Chlamydomonas axonemal inner dynein arm I1. Structural comparisons reveal a strong similarity with the LC8 dynein light chain dimer, including formation of a strand-switched beta sheet interface. Analysis of the Tctex1 structure enables the dynein intermediate chain binding site to be identified and suggests a mechanism by which cargo proteins might be attached to this microtubule motor complex. Comparison with the alternate dynein light chain rp3 reveals how the specificity of dynein-cargo interactions mediated by these dynein components is achieved. In addition, this structure provides insight into the consequences of the mutations found in the t haplotype forms of this protein.