Cytoplasmic Dynein Light Intermediate Chain Research Articles

Tight coupling between nucleus and cell migration through the perinuclear actin cap

Although eukaryotic cells are known to alternate between 'advancing' episodes of fast and persistent movement and 'hesitation' episodes of low speed and low persistence, the molecular mechanism that controls the dynamic changes in morphology, speed and persistence of eukaryotic migratory cells remains unclear. Here, we show that the movement of the interphase nucleus during random cell migration switches intermittently between two distinct modes - rotation and translocation - that follow with high fidelity the sequential rounded and elongated morphologies of the nucleus and cell body, respectively. Nuclear rotation and translocation mediate the stop-and-go motion of the cell through the dynamic formation and dissolution, respectively, of the contractile perinuclear actin cap, which is dynamically coupled to the nuclear lamina and the nuclear envelope through LINC complexes. A persistent cell movement and nuclear translocation driven by the actin cap are halted following the disruption of the actin cap, which in turn allows the cell to repolarize for its next persistent move owing to nuclear rotation mediated by cytoplasmic dynein light intermediate chain 2.

Behavioral and other phenotypes in a cytoplasmic Dynein light intermediate chain 1 mutant mouse.

The cytoplasmic dynein complex is fundamentally important to all eukaryotic cells for transporting a variety of essential cargoes along microtubules within the cell. This complex also plays more specialized roles in neurons. The complex consists of 11 types of protein that interact with each other and with external adaptors, regulators and cargoes. Despite the importance of the cytoplasmic dynein complex, we know comparatively little of the roles of each component protein, and in mammals few mutants exist that allow us to explore the effects of defects in dynein-controlled processes in the context of the whole organism. Here we have taken a genotype-driven approach in mouse (Mus musculus) to analyze the role of one subunit, the dynein light intermediate chain 1 (Dync1li1). We find that, surprisingly, an N235Y point mutation in this protein results in altered neuronal development, as shown from in vivo studies in the developing cortex, and analyses of electrophysiological function. Moreover, mutant mice display increased anxiety, thus linking dynein functions to a behavioral phenotype in mammals for the first time. These results demonstrate the important role that dynein-controlled processes play in the correct development and function of the mammalian nervous system.

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.

Identification of genes involved in the ciliary trafficking of C. elegans PKD‐2

Ciliary membrane proteins are important extracellular sensors, and defects in their localization may have profound developmental and physiological consequences. To determine how sensory receptors localize to cilia, we performed a forward genetic screen and identified 11 mutants with defects in the ciliary localization (cil) of C. elegans PKD-2, a transient receptor potential polycystin (TRPP) channel. Class A cil mutants exhibit defects in PKD-2::GFP somatodendritic localization while Class B cil mutants abnormally accumulate PKD-2::GFP in cilia. Further characterization reveals that some genes mutated in cil mutants act in a tissue-specific manner while others are likely to play more general roles in such processes as intraflagellar transport (IFT). To this end, we identified a Class B mutation that disrupts the function of the cytoplasmic dynein light intermediate chain gene xbx-1. Identification of the remaining mutations will reveal novel molecular pathways required for ciliary receptor localization and provide further insight into mechanisms of ciliary signaling.

Cytoplasmic dynein light intermediate chain is required for discrete aspects of mitosis in Caenorhabditis elegans.

We describe phenotypic characterization of dli-1, the Caenorhabditis elegans homolog of cytoplasmic dynein light intermediate chain (LIC), a subunit of the cytoplasmic dynein motor complex. Animals homozygous for loss-of-function mutations in dli-1 exhibit stochastic failed divisions in late larval cell lineages, resulting in zygotic sterility. dli-1 is required for dynein function during mitosis. Depletion of the dli-1 gene product through RNA-mediated gene interference (RNAi) reveals an early embryonic requirement. One-cell dli-1(RNAi) embryos exhibit failed cell division attempts, resulting from a variety of mitotic defects. Specifically, pronuclear migration, centrosome separation, and centrosome association with the male pronuclear envelope are defective in dli-1(RNAi) embryos. Meiotic spindle formation, however, is not affected in these embryos. DLI-1, like its vertebrate homologs, contains a putative nucleotide-binding domain similar to those found in the ATP-binding cassette transporter family of ATPases as well as other nucleotide-binding and -hydrolyzing proteins. Amino acid substitutions in a conserved lysine residue, known to be required for nucleotide binding, confers complete rescue in a dli-1 mutant background, indicating this is not an essential domain for DLI-1 function.

Direct interaction of pericentrin with cytoplasmic dynein light intermediate chain contributes to mitotic spindle organization.

Pericentrin is a conserved protein of the centrosome involved in microtubule organization. To better understand pericentrin function, we overexpressed the protein in somatic cells and assayed for changes in the composition and function of mitotic spindles and spindle poles. Spindles in pericentrin-overexpressing cells were disorganized and mispositioned, and chromosomes were misaligned and missegregated during cell division, giving rise to aneuploid cells. We unexpectedly found that levels of the molecular motor cytoplasmic dynein were dramatically reduced at spindle poles. Cytoplasmic dynein was diminished at kinetochores also, and the dynein-mediated organization of the Golgi complex was disrupted. Dynein coimmunoprecipitated with overexpressed pericentrin, suggesting that the motor was sequestered in the cytoplasm and was prevented from associating with its cellular targets. Immunoprecipitation of endogenous pericentrin also pulled down cytoplasmic dynein in untransfected cells. To define the basis for this interaction, pericentrin was coexpressed with cytoplasmic dynein heavy (DHCs), intermediate (DICs), and light intermediate (LICs) chains, and the dynamitin and p150(Glued) subunits of dynactin. Only the LICs coimmunoprecipitated with pericentrin. These results provide the first physiological role for LIC, and they suggest that a pericentrin-dynein interaction in vivo contributes to the assembly, organization, and function of centrosomes and mitotic spindles.

Characterization of cytoplasmic dynein light-intermediate chain isoforms in rat testis.

We obtained three kinds of novel cytoplasmic dynein light-intermediate chain (LIC) isoforms from rat testis and brain by reverse transcription polymerase chain reaction (RT-PCR). The primers for RT-PCR were designed according to LIC-2 (LIC 53/55) (15). In one novel isoform, the 42 bp specific sequence named TDL was inserted between 1,106 and 1,107 nucleotides (nts) of LIC-2, whereas the 57 bp sequence corresponding to LIC-2 1,339-1,395 nts (BDL) was absent. The TDL and BDL regions were specifically digested with restriction enzymatic treatment and followed by subcloning of non-digested cDNA band in testis and brain, producing an isoform without TDL and BDL regions. BDL specific RT-PCR of testis cDNA followed by sequencing produced an isoform with two specific regions. By Northern blot hybridization using TDL and BDL specific antisense oligo DNA probe, 4.4, 3.5, and 2.0 kb of signals were detected. With both TDL and BDL probes, the 2.0 kb signal was intensely detected in testis, while the 4.4 kb was defected in brain. This indicates that TDL and BDL are derived from the same size of mRNAs. In situ hybridization method using these probes showed that all seminiferous epithelial cells, especially late pachytene spermatocytes, were positive, indicating that LIC 53/55 isoforms were coexpressed in these cells. These findings indicate that LIC 53/55 isoforms provide a variety of dynein subunits, and thus may regulate the dynein-dependent intracellular transport system.

Localization of Cytoplasmic Dynein Light-intermediate Chain mRNA in the Rat Testis Using In Situ Hybridization.

Expression of cytoplasmic dynein light-intermediate chain mRNA in the rat testis was examined using in situ hybridization. The ribonucleotide probe, referred to the 5' end of open reading frame (6-515 nucleotids) of cytoplasmic dynein light-intermediate chain 53/55 (LIC-2) of the rat brain (Hughes et al., 1995. J. Cell Sci., 108: 17-24), was used. All spermatogenic cells were positive. Pachytene spermatocytes in later stages (after-stage VII) were the most intensely positive and round spermatids were also intense. These findings indicated that all spermatogenic cells may store the light-intermediate chain signal, and spermatocytes may produce it during later stages. The reaction in Sertoli cells was constant in intensity during the spermatogenic cycle, indicating that the light-intermediate chain mRNA signal may have no relation to the stage-dependent organelle transport, and that there may be post-translational regulation of the light-intermediate chain. In interstitium, only a few positive cells were observed. Northern blot hybridization demonstrated that one major band (2.0 kb) and two minor bands (4.4 kb and 3.5 kb) were detected in the testis, while one major band (4.4 kb) and one minor band (3.5 kb) were in the brain. This indicated that there are at least 3 isoforms in cytoplasmic dynein light-intermediate chain 53/55.

Molecular analysis of a cytoplasmic dynein light intermediate chain reveals homology to a family of ATPases

Cytoplasmic dynein is a multi-subunit complex involved in retrograde organelle transport and some aspects of mitosis. In previous work we have cloned and sequenced cDNAs encoding the rat cytoplasmic dynein heavy and intermediate chains. Here we report the cloning of the remaining class of cytoplasmic dynein subunits, which we refer to as the light intermediate chains (LICs: 53-59 kDa). Four LIC electrophoretic bands were resolved in purified bovine cytoplasmic dynein preparations by one-dimensional gel electrophoresis. These four bands were simplified to two bands (LIC53/55 and LIC57/59) by alkaline phosphatase treatment. N-terminal amino acid sequence was obtained from a total of 11 proteolytic peptides generated from both LIC53/55 and LIC57/59. Overlapping cDNA clones encoding LIC53/55 were isolated by oligonucleotide screening using probes based on the LIC53/55 peptide sequence. The cDNA sequence contained a 497 codon open reading frame encoding a polypeptide with a molecular mass of approximately 55 kDa. Each of the LIC53/55 peptides was found within the deduced amino acid sequence, as well as four of the LIC57/59 peptides. Analysis of the LIC53/55 primary sequence revealed homology with the ABC transporter family of ATPases in the region surrounding the P-loop sequence element. Together these data identify the LICs as a novel family of dynein subunits with potential ATPase activity. They also reveal that the complexity of the LICs is due to both post-translational modification and the existence of at least two LIC polypeptides for which we propose the names LIC-1a and LIC-2.