Dynein LIC1 localizes to the mitotic spindle and midbody and LIC2 localizes to spindle poles during cell division

CD-1 (cytoplasmic dynein-1) is a multisubunit motor protein complex involved in intracellular trafficking and mitosis. The dynein LIC (light intermediate chain) subunits, LIC1 (DLIC-1, gene symbol DYNC1LI1) and LIC2 (DLIC-2, gene symbol DYNC1LI2), associate with the dynein HC (heavy chain) in a mutually exclusive manner and thus define distinct functional CD-1 complexes. Here, we analysed the mitotic distribution of LIC1 and LIC2. We found that from metaphase through anaphase, LIC1 localizes to the mitotic spindle and concentrates within the midbody during the abscission step of cytokinesis. Conversely, LIC2 strongly localizes to the spindle poles from prophase through telophase. These data suggest distinct functions for LIC1 and LIC2-containing CD-1 complexes during cell division.     

Mutations in the Gene Encoding IFT Dynein Complex Component WDR34 Cause Jeune Asphyxiating Thoracic Dystrophy

Bidirectional (anterograde and retrograde) motor-based intraflagellar transport (IFT) governs cargo transport and delivery processes that are essential for primary cilia growth and maintenance and for hedgehog signaling functions. The IFT dynein-2 motor complex that regulates ciliary retrograde protein transport contains a heavy chain dynein ATPase/motor subunit, DYNC2H1, along with other less well functionally defined subunits. Deficiency of IFT proteins, including DYNC2H1, underlies a spectrum of skeletal ciliopathies. Here, by using exome sequencing and a targeted next-generation sequencing panel, we identified a total of 11 mutations in WDR34 in 9 families with the clinical diagnosis of Jeune syndrome (asphyxiating thoracic dystrophy). WDR34 encodes a WD40 repeat-containing protein orthologous to Chlamydomonas FAP133, a dynein intermediate chain associated with the retrograde intraflagellar transport motor. Three-dimensional protein modeling suggests that the identified mutations all affect residues critical for WDR34 protein-protein interactions. We find that WDR34 concentrates around the centrioles and basal bodies in mammalian cells, also showing axonemal staining. WDR34 coimmunoprecipitates with the dynein-1 light chain DYNLL1 in vitro, and mining of proteomics data suggests that WDR34 could represent a previously unrecognized link between the cytoplasmic dynein-1 and IFT dynein-2 motors. Together, these data show that WDR34 is critical for ciliary functions essential to normal development and survival, most probably as a previously unrecognized component of the mammalian dynein-IFT machinery.     

Class I Rab11‐family interacting proteins are binding targets for the Rab14 GTPase

Background information. Rab11 and Rab14 are two related Rab GTPases that are believed to function in endosomal recycling and Golgi/endosome transport processes. We, and others, have identified a group of proteins that interact with Rab11 and function as Rab11 effectors, known as the Rab11‐FIPs (family interacting proteins). This protein family has been sub‐classified into two groups—class I FIPs [FIP2, RCP (Rab coupling protein) and Rip11 (Rab11‐interacting protein)] and class II FIPs (FIP3 and FIP4). Results. In the present study we identify the class I FIPs as dual Rab‐binding proteins by demonstrating that they also interact with Rab14 in a GTP‐dependent manner. We show that these interactions are specific for the class I FIPs and that they occur via their C‐terminal regions, which encompass the previously described RBD (Rab11‐binding domain). Furthermore, we show that Rab14 significantly co‐localizes with the TfnR (transferrin receptor) and that Rab14 Q70L co‐localizes with Rab11a and with the class I FIPs on the ERC (endosomal recycling compartment) during interphase. Additionally, we show that during cytokinesis Rab14 localizes to the cleavage furrow/midbody. Conclusions. The data presented in the present study, which identifies the class I FIPs as the first putative effector proteins for the Rab14 GTPase, indicates greater complexity in the Rab‐binding specificity of the class I FIP proteins.     

HCMV-Encoded Glycoprotein M (UL100) Interacts with Rab11 Effector Protein FIP4

The envelope of human cytomegalovirus (HCMV) consists of a large number of glycoproteins. The most abundant glycoprotein in the HCMV envelope is the glycoprotein M (UL100), which together with glycoprotein N (UL73) form the gM/gN protein complex. Using yeast two-hybrid screening, we found that the gM carboxy-terminal cytoplasmic tail (gM-CT) interacts with FIP4, a Rab11-GTPase effector protein. Depletion of FIP4 expression in HCMV-infected cells resulted in a decrease in infectious virus production that was also associated with an alteration of the HCMV assembly compartment (AC) phenotype. A similar phenotype was also observed in HCMV-infected cells that expressed dominant negative Rab11(S25N). Recently, it has been shown that FIP4 interactions with Rab11 and additionally with Arf6/Arf5 are important for the vesicular transport of proteins in the endosomal recycling compartment (ERC) and during cytokinesis. Surprisingly, FIP4 interaction with gM-CT limited binding of FIP4 with Arf5/Arf6; however, FIP4 interaction with gM-CT did not prevent recruitment of Rab11 into the ternary complex. These data argued for a contribution of the ERC during cytoplasmic envelopment of HCMV and showed a novel FIP4 function independent of Arf5 or Arf6 activity.     

Molecular characterization of Rab11-FIP3 binding to ARF GTPases

Rab11-FIP3 is a Rab11-binding protein that has been implicated in regulating cytokinesis in mammalian cells. FIP3 functions by simultaneously interacting with Rab11 as well as Arf GTPases. However, unlike the interaction between Rab11 and FIP3, the structural basis of FIP3 binding to Arf GTPases has not yet been determined. The specificity of interaction between FIP3 and Arf GTPases remains controversial. While it was reported that FIP3 preferentially binds to Arf6 some data suggest that FIP3 can also interact with Arf5 and even possibly Arf4. The Arf-interaction motif on FIP3 also remains to be determined. Finally, the importance of Arf binding to FIP3 in regulating cell division and other cellular functions remains unclear. Here we used a combination of various biochemical techniques to measure the affinity of FIP3 binding to various Arfs and to demonstrate that FIP3 predominantly interacts with Arf6 in vitro and in vivo. In addition, we identified the motifs mediating Arf6 and FIP3 interaction and demonstrated that FIP3 binds to the Arf6 C-terminus rather than switch motifs. Finally we show that FIP3 and Arf6 binding is required for the targeting of Arf6 to the cleavage furrow during cytokinesis. Thus, we propose that FIP3 is a scaffolding protein that, in addition to regulating endosome targeting to the cleavage furrow, also is required for Arf6 recruitment to the midbody during late telophase.     

Rab11‐FIP2 Interaction with MYO5B Regulates Movement of Rab11a‐Containing Recycling Vesicles

A tripartite association of Rab11a with both Rab11‐FIP2 and MYO5B regulates recycling endosome trafficking. We sought to define the intermolecular interactions required between Rab11‐FIP2 and MYO5B. Using a random mutagenesis strategy, we identified point mutations at S229P or G233E in Rab11‐FIP2 that caused loss of interaction with MYO5B in yeast two‐hybrid assays as well as loss of interaction of Rab11‐FIP2(129‐356) with MYO5B tail when expressed in HeLa cells. Single mutations or the double S229P/G233E mutation failed to alter the association of full‐length Rab11‐FIP2 with MYO5B tail in HeLa cells. While EGFP‐Rab11‐FIP2 wild type colocalized with endogenous MYO5B staining in MDCK cells, EGFP‐Rab11‐FIP2(S229P/G233E) showed a significant decrease in localization with endogenous MYO5B. Analysis of Rab11a‐containing vesicle movement in live HeLa cells demonstrated that when the MYO5B/Rab11‐FIP2 association is perturbed by mutation or by Rab11‐FIP2 knockdown, vesicle movement is increased in both speed and track length, consistent with an impairment of MYO5B tethering at the cytoskeleton. These results support a critical role for the interaction of MYO5B with Rab11‐FIP2 in stabilizing the functional complex with Rab11a, which regulates dynamic movements of membrane recycling vesicles.      

A role for Tctex-1 (DYNLT1) in controlling primary cilium length

The microtubule motor complex cytoplasmic dynein is known to be involved in multiple processes including endomembrane organization and trafficking, mitosis, and microtubule organization. The majority of studies of cytoplasmic dynein have focused on the form of the motor that is built around the dynein-1 heavy chain. A second isoform, dynein heavy chain-2, and its specifically associated light intermediate chain, LIC3 (D2LIC), are known to be involved in the formation and function of primary cilia. We have used RNAi in human epithelial cells to define the cytoplasmic dynein subunits that function with dynein heavy chain 2 in primary cilia. We identify the dynein light chain Tctex-1 as a key modulator of cilia length control; depletion of Tctex-1 results in longer cilia as defined by both acetylated tubulin labeling of the axoneme and Rab8a labeling of the cilia membrane. Suppression of dynein heavy chain-2 causes concomitant loss of Tctex-1 and this correlates with an increase in cilia length. Compared to individual depletions, double siRNA depletion of DHC2 and Tctex-1 causes an even greater increase in cilia length. Our data show that Tctex-1 is a key regulator of cilia length and most likely functions as part of dynein-2.     

Dynein binds and stimulates axonal motility of the endosome adaptor and NEEP21 family member,calcyon

The neuron-enriched, endosomal protein Calcyon (Caly) regulates endocytosis and vesicle sorting, and is important for synaptic plasticity and brain development. In the current investigation of Caly interacting proteins in brain, the microtubule retrograde motor subunit, cytoplasmic dynein 1 heavy chain (DYNC1H), and microtubule structural proteins, α and β tubulin, were identified as Caly associated proteins by MALDI-ToF/ToF. Direct interaction of the Caly-C terminus with dynein and tubulin was further confirmed in in vitro studies. In Cos-7 cells, mCherry-Caly moved along the microtubule network in organelles largely labeled by the late endosome marker Rab7. Expression of the dynein inhibitor CC1, produced striking alterations in Caly distribution, consistent with retrograde motors playing a prominent role in Caly localization and movement. In axons of cultured adult rat sensory neurons, Caly-positive organelles co-localized with dynein intermediate chain (DYNC1I1-isoform IC-1B) and the dynein regulator, lissencephaly 1 (LIS1), both of which co-precipitated from brain with the Caly C-terminus. Manipulation of dynein function in axons altered the motile properties of Caly indicating that Caly vesicles utilize the retrograde motor. Altogether, the current evidence for association with dynein motors raises the possibility that the endocytic and cargo sorting functions of Caly in neurons could be regulated by interaction with the microtubule transport system.     

Rab11-FIP3 is critical for the structural integrity of the endosomal recycling compartment

Rab11-FIP3 is an endosomal recycling compartment (ERC) protein that is implicated in the process of membrane delivery from the ERC to sites of membrane insertion during cell division. Here we report that Rab11-FIP3 is critical for the structural integrity of the ERC during interphase. We demonstrate that knockdown of Rab11-FIP3 and expression of a mutant of Rab11-FIP3 that is Rab11-binding deficient cause loss of all ERC-marker protein staining from the pericentrosomal region of A431 cells. Furthermore, we find that fluorophore-labelled transferrin cannot access the pericentrosomal region of cells in which Rab11-FIP3 function has been perturbed. We find that this Rab11-FIP3 function appears to be specific because expression of the equivalent Rab11-binding deficient mutant of Rab-coupling protein does not perturb ERC morphology. In addition, we find that other organelles such as sorting and late endosomes are unaffected by loss of Rab11-FIP3 function. Finally, we demonstrate the presence of an extensive coiled-coil region between residues 463 and 692 of Rab11-FIP3, which exists as a dimer in solution and is critical to support its function on the ERC. Together, these data indicate that Rab11-FIP3 is necessary for the structural integrity of the pericentrosomal ERC.     

Recruitment of dynein to late endosomes and lysosomes through light intermediate chains

Cytoplasmic dynein is involved in a wide range of cellular processes, but how it is regulated and how it recognizes an extremely wide range of cargo are incompletely understood. The dynein light intermediate chains, LIC1 and LIC2 (DYNC1LI1 and DYNC1LI2, respectively), have been implicated in cargo binding, but their full range of functions is unknown. Using LIC isoform-specific antibodies, we report the first characterization of their subcellular distribution and identify a specific association with elements of the late endocytic pathway, but not other vesicular compartments. LIC1 and LIC2 RNA interference (RNAi) each specifically disrupts the distribution of lysosomes and late endosomes. Stimulation of dynein-mediated late-endosomal transport by the Rab7-interacting lysosomal protein (RILP) is reversed by LIC1 RNAi, which displaces dynein, but not dynactin, from these structures. Conversely, expression of ΔN-RILP or the dynactin subunit dynamitin each fails to displace dynein, but not dynactin. Thus, using a variety of complementary approaches, our results indicate a novel specific role for the LICs in dynein recruitment to components of the late endocytic pathway.     

An InCytes from MBC Selection: Specificity of Cytoplasmic Dynein Subunits in Discrete Membrane-trafficking Steps

The cytoplasmic dynein motor complex is known to exist in multiple forms, but few specific functions have been assigned to individual subunits. A key limitation in the analysis of dynein in intact mammalian cells has been the reliance on gross perturbation of dynein function, e.g., inhibitory antibodies, depolymerization of the entire microtubule network, or the use of expression of dominant negative proteins that inhibit dynein indirectly. Here, we have used RNAi and automated image analysis to define roles for dynein subunits in distinct membrane-trafficking processes. Depletion of a specific subset of dynein subunits, notably LIC1 (DYNC1LI1) but not LIC2 (DYNC1LI2), recapitulates a direct block of ER export, revealing that dynein is required to maintain the steady-state composition of the Golgi, through ongoing ER-to-Golgi transport. Suppression of LIC2 but not of LIC1 results in a defect in recycling endosome distribution and cytokinesis. Biochemical analyses also define the role of each subunit in stabilization of the dynein complex; notably, suppression of DHC1 or IC2 results in concomitant loss of Tctex1. Our data demonstrate that LIC1 and LIC2 define distinct dynein complexes that function at the Golgi versus recycling endosomes, respectively, suggesting that functional populations of dynein mediate discrete intracellular trafficking pathways.     

Cytoplasmic dynein-2: from molecules to human diseases

The dynein motor protein family is involved in a wide variety of functions in eukaryotic cells. The axonemal dynein class and cytoplasmic dynein-1 subclass have been well characterized. However, the cytoplasmic dynein-2 subclass of the family has only recently begun to be understood. We describe the entire dynein family but focus on cytoplasmic dynein-2. Dynein-2 consists of a heavy, an intermediate, a light intermediate, and a light chain. The complex appears to function primarily as the retrograde motor for intraflagellar transport. This process is important for the formation and maintenance of cilia and flagella. Additionally, dynein-2 has roles in the control of ciliary length and in non-ciliary functions. Mutations in the human dynein-2 heavy chain lead to cilia-related diseases.     

Recombinant human cytoplasmic dynein heavy chain 1 and 2: observation of dynein-2 motor activity in vitro

Cytoplasmic dynein is a microtubule (MT) motor protein comprising two classes: dynein-1 and dynein-2. We purified recombinant human dynein-1 and dynein-2 from HEK-293 cells by expressing the streptavidin-binding peptide-tagged human cytoplasmic dynein-1 and dynein-2 heavy chains (HCs), respectively. Electron microscopy of the purified molecules revealed a two-headed structure composed of characteristic dynein motor domains. In an in vitro MT gliding assay, both dynein-1 and dynein-2 showed minus-end-directed motor activities. This is the first demonstration of dynein-2 motor activity, which supports the retrograde intraflagellar transport role of dynein-2. Our expression system of dynein HCs provides a useful means to investigate dynein functions.     

A Highlights from MBoC Selection: Evidence for dynein and astral microtubule–mediated cortical release and transport of Gαi/LGN/NuMA complex in mitotic cells

Spindle positioning is believed to be governed by the interaction between astral microtubules and the cell cortex and involve cortically anchored motor protein dynein. How dynein is recruited to and regulated at the cell cortex to generate forces on astral microtubules is not clear. Here we show that mammalian homologue of Drosophila Pins (Partner of Inscuteable) (LGN), a Gα_i-binding protein that is critical for spindle positioning in different systems, associates with cytoplasmic dynein heavy chain (DYNC1H1) in a Gα_i-regulated manner. LGN is required for the mitotic cortical localization of DYNC1H1, which, in turn, also modulates the cortical accumulation of LGN. Using fluorescence recovery after photobleaching analysis, we show that cortical LGN is dynamic and the turnover of LGN relies, at least partially, on astral microtubules and DYNC1H1. We provide evidence for dynein- and astral microtubule–mediated transport of Gα_i/LGN/nuclear mitotic apparatus (NuMA) complex from cell cortex to spindle poles and show that actin filaments counteract such transport by maintaining Gα_i/LGN/NuMA and dynein at the cell cortex. Our results indicate that astral microtubules are required for establishing bipolar, symmetrical cortical LGN distribution during metaphase. We propose that regulated cortical release and transport of LGN complex along astral microtubules may contribute to spindle positioning in mammalian cells.     

The Rab11-family interacting proteins reveal selective interaction of mammalian recycling endosomes with the Toxoplasma parasitophorous vacuole in a Rab11- and Arf6-dependent manner

After mammalian cell invasion, the parasite Toxoplasma multiplies in a self-made membrane-bound compartment, the parasitophorous vacuole (PV). We previously showed that Toxoplasma interacts with many host cell organelles, especially from recycling pathways, and sequestrates Rab11A and Rab11B vesicles into the PV. Here, we examine the specificity of host Rab11 vesicle interaction with the PV by focusing on the recruitment of subpopulations of Rab11 vesicles characterized by different effectors, for example, Rab11-family interacting roteins (FIPs) or Arf6. Our quantitative microscopic analysis illustrates the presence of intra-PV vesicles with FIPs from class I (FIP1C, FIP2, FIP5) and class II (FIP3, FIP4) but to various degrees. The intra-PV delivery of vesicles with class I, but not class II, FIPs is dependent on Rab11 binding. Cell depletion of Rab11A results in a significant decrease in intra-PV FIP5, but not FIP3 vesicles. Class II FIPs also bind to Arf6, and we observe vesicles associated with FIP3-Rab11A or FIP3-Arf6 complexes concomitantly within the PV. Abolishing FIP3 binding to both Rab11 and Arf6 reduces the number of intra-PV FIP3 vesicles. These data point to a selective process of mammalian Rab11 vesicle recognition and scavenging mediated by Toxoplasma, suggesting that specific parasite PV proteins may be involved in these processes.     

FIP2 and Rip11 specify Rab11a-mediated cellular distribution of GLUT4 and FAT/CD36 in H9c2-hIR cells

Rab11a has been shown to be involved in different vesicle trafficking processes. To further define the functional role of Rab11a in vesicle movement we knocked down gene expression of Rab11a and two of its effectors, Rip11 and FIP2, in H9c2-hIR cells and measured the cell surface abundance of GLUT4myc and FAT/CD36. We observed that by knocking down Rab11a, both GLUT4myc and FAT/CD36 abundance at the plasma membrane were substantially increased. In the case of GLUT4myc, the in vitro knockdown of FIP2 also increased the cell surface abundance of GLUT4myc. Knockdown of both FIP2 and Rip11 increase the abundance of FAT/CD36 at the plasma membrane. Stimulated translocation of GLUT4myc and FAT/CD36 is not altered after gene knockdown of Rab11a. These data therefore show that (i) Rab11a regulates cell surface abundance of both GLUT4 and FAT/CD36 and that (ii) both Rab11a-dependent processes are differently regulated by Rab11a effector proteins.     

Dynein light intermediate chains maintain spindle bipolarity by functioning in centriole cohesion

Cytoplasmic dynein 1 (dynein) is a minus end–directed microtubule motor protein with many cellular functions, including during cell division. The role of the light intermediate chains (LICs; DYNC1LI1 and 2) within the complex is poorly understood. In this paper, we have used small interfering RNAs or morpholino oligonucleotides to deplete the LICs in human cell lines and Xenopus laevis early embryos to dissect the LICs’ role in cell division. We show that although dynein lacking LICs drives microtubule gliding at normal rates, the LICs are required for the formation and maintenance of a bipolar spindle. Multipolar spindles with poles that contain single centrioles were formed in cells lacking LICs, indicating that they are needed for maintaining centrosome integrity. The formation of multipolar spindles via centrosome splitting after LIC depletion could be rescued by inhibiting Eg5. This suggests a novel role for the dynein complex, counteracted by Eg5, in the maintenance of centriole cohesion during mitosis.