Regulatory dissociation of Tctex-1 light chain from dynein complex is essential for the apical delivery of rhodopsin

Post-Golgi to apical surface delivery in polarized epithelial cells requires the cytoplasmic dynein motor complex. However, the nature of dynein-cargo interactions and their underlying regulation are largely unknown. Previous studies have shown that the apical surface targeting of rhodopsin requires the dynein light chain, Tctex-1, which binds directly to both dynein intermediate chain (IC) and rhodopsin. In this report, we show that the S82E mutant of Tctex-1, which mimics Tctex-1 phosphorylated at serine 82, has a reduced affinity for dynein IC but not for rhodopsin. Velocity sedimentation experiments further suggest that S82E is not incorporated into the dynein complex. The dominant-negative effect of S82E causes rhodopsin mislocalization in polarized Madin-Darby canine kidney (MDCK) cells. The S82A mutant, which mimics dephosphorylated Tctex-1, can be incorporated into dynein complex but is impaired in its release. Expression of S82A also causes disruption of the apical localization of rhodopsin in MDCK cells. Taken together, these results suggest that the dynein complex disassembles to release cargo due to the specific phosphorylation of Tctex-1 at the S82 residue and that this process is critical for the apical delivery of membrane cargoes.     

Cytoplasmic dynein/dynactin drives kinetochore protein transport to the spindle poles and has a role in mitotic spindle checkpoint inactivation.

We discovered that many proteins located in the kinetochore outer domain, but not the inner core, are depleted from kinetochores and accumulate at spindle poles when ATP production is suppressed in PtK1 cells, and that microtubule depolymerization inhibits this process. These proteins include the microtubule motors CENP-E and cytoplasmic dynein, and proteins involved with the mitotic spindle checkpoint, Mad2, Bub1R, and the 3F3/2 phosphoantigen. Depletion of these components did not disrupt kinetochore outer domain structure or alter metaphase kinetochore microtubule number. Inhibition of dynein/dynactin activity by microinjection in prometaphase with purified p50 "dynamitin" protein or concentrated 70.1 anti-dynein antibody blocked outer domain protein transport to the spindle poles, prevented Mad2 depletion from kinetochores despite normal kinetochore microtubule numbers, reduced metaphase kinetochore tension by 40%, and induced a mitotic block at metaphase. Dynein/dynactin inhibition did not block chromosome congression to the spindle equator in prometaphase, or segregation to the poles in anaphase when the spindle checkpoint was inactivated by microinjection with Mad2 antibodies. Thus, a major function of dynein/dynactin in mitosis is in a kinetochore disassembly pathway that contributes to inactivation of the spindle checkpoint.     

CRMP-2 directly binds to cytoplasmic dynein and interferes with its activity

The active transport of proteins and organelles is critical for cellular organization and function in eukaryotic cells. A substantial portion of long-distance transport depends on the opposite polarity of the kinesin and dynein family molecular motors to move cargo along microtubules. It is increasingly clear that many cargo molecules are moved bi-directionally by both sets of motors; however, the regulatory mechanism that determines the directionality of transport remains unclear. We previously reported that collapsin response mediator protein-2 (CRMP-2) played key roles in axon elongation and neuronal polarization. CRMP-2 was also found to associate with the anterograde motor protein Kinesin-1 and was transported with other cargoes toward the axon terminal. In this study, we investigated the association of CRMP-2 with a retrograde motor protein, cytoplasmic dynein. Immunoprecipitation assays showed that CRMP-2 interacted with cytoplasmic dynein heavy chain. Dynein heavy chain directly bound to the N-terminus of CRMP-2, which is the distinct side of CRMP-2's kinesin light chain-binding region. Furthermore, over-expression of the dynein-binding fragments of CRMP-2 prevented dynein-driven microtubule transport in COS-7 cells. Given that CRMP-2 is a key regulator of axon elongation, this interference with cytoplasmic dynein function by CRMP-2 might have an important role in axon formation, and neuronal development.     

Cytoplasmic dynein: a key player in neurodegenerative and neurodevelopmental diseases

Cytoplasmic dynein is the most important molecular motor driving the movement of a wide range of cargoes towards the minus ends of microtubules.As a molecular motor protein,dynein performs a variety of basic cellular functions including organelle transport and centrosome assembly.In the nervous system,dynein has been demonstrated to be responsible for axonal retrograde transport.Many studies have revealed direct or indirect evidence of dynein in neurodegenerative diseases such as amyotrophic lateral sclerosis,Charcot-Marie-Tooth disease,Alzheimer’s disease,Parkinson’s disease and Huntington’s disease.Among them,a number of mutant proteins involved in various neurodegenerative diseases interact with dynein.Axonal transport disruption is presented as a common feature occurring in neurodegenerative diseases.Dynein heavy chain mutant mice also show features of neurodegenerative diseases.Moreover,defects of dynein-dependent processes such as autophagy or clearance of aggregation-prone proteins are found in most of these diseases.Lines of evidence have also shown that dynein is associated with neurodevelopmental diseases.In this review,we focus on dynein involvement in different neurological diseases and discuss potential underlying mechanisms.     

GSK‐3β Phosphorylation of Cytoplasmic Dynein Reduces Ndel1 Binding to Intermediate Chains and Alters Dynein Motility

Glycogen synthase kinase 3 (GSK‐3) has been linked to regulation of kinesin‐dependent axonal transport in squid and flies, and to indirect regulation of cytoplasmic dynein. We have now found evidence for direct regulation of dynein by mammalian GSK‐3β in both neurons and non‐neuronal cells. GSK‐3β coprecipitates with and phosphorylates mammalian dynein. Phosphorylation of dynein intermediate chain (IC) reduces its interaction with Ndel1, a protein that contributes to dynein force generation. Two conserved residues, S87/T88 in IC‐1B and S88/T89 in IC‐2C, have been identified as GSK‐3 targets by both mass spectrometry and site‐directed mutagenesis. These sites are within an Ndel1‐binding domain, and mutation of both sites alters the interaction of IC's with Ndel1. Dynein motility is stimulated by (i) pharmacological and genetic inhibition of GSK‐3β, (ii) an insulin‐sensitizing agent (rosiglitazone) and (iii) manipulating an insulin response pathway that leads to GSK‐3β inactivation. Thus, our study connects a well‐characterized insulin‐signaling pathway directly to dynein stimulation via GSK‐3 inhibition.      

The established and the predicted roles of dynein light chain in the regulation of mitochondrial apoptosis

The mitochondrial pathway of apoptosis is regulated by the interplay between the members of Bcl-2 family. Within this family, BH3-only proteins are the sensors of apoptotic stimuli and can trigger apoptosis either by inhibiting the anti-apoptotic Bcl-2-family proteins or by directly activating the effectors Bax and Bak. An expanding body of research suggests that a number of non-Bcl-2 proteins can also interact with Bcl-2 proteins and contribute to the decision of cell fate. Dynein light chain (LC8, DYNLL or DLC), a hub protein and a dimerizing engine has been proposed to regulate the pro-apoptotic activity of two BH3-only proteins, Bim and Bmf. Our recent work has provided insight into the mechanisms through which DLC1 (DYNLL1) modulates Bim activity. Here we discuss the present day understanding of Bim-DLC interaction and endeavor to evaluate this interaction in the light of information from studies of DLC with other binding partners.     

Cell-nonautonomous inhibition of radiation-induced apoptosis by dynein light chain 1 in Caenorhabditis elegans

The evolutionarily conserved process of programmed cell death, apoptosis, is essential for development of multicellular organisms and is also a protective mechanism against cellular damage. We have identified dynein light chain 1 (DLC-1) as a new regulator of germ cell apoptosis in Caenorhabditis elegans. The DLC-1 protein is highly conserved across species and is a part of the dynein motor complex. There is, however, increasing evidence for dynein-independent functions of DLC-1, and our data describe a novel dynein-independent role. In mammalian cells, DLC-1 is important for cellular transport, cell division and regulation of protein activity, and it has been implicated in cancer. In C. elegans, we find that knockdown of dlc-1 by RNA interference (RNAi) induces excessive apoptosis in the germline but not in somatic cells during development. We show that DLC-1 mediates apoptosis through the genes lin-35, egl-1 and ced-13, which are all involved in the response to ionising radiation (IR)-induced apoptosis. In accordance with this, we show that IR cannot further induce apoptosis in dlc-1(RNAi) animals. Furthermore, we find that DLC-1 is functioning cell nonautonomously through the same pathway as kri-1 in response to IR-induced apoptosis and that DLC-1 regulates the levels of KRI-1. Our results strengthen the notion of a highly dynamic communication between somatic cells and germ cells in regulating the apoptotic process.     

Load‐dependent detachment kinetics plays a key role in bidirectional cargo transport by kinesin and dynein

Bidirectional cargo transport along microtubules is carried out by opposing teams of kinesin and dynein motors. Despite considerable study, the factors that determine whether these competing teams achieve net anterograde or retrograde transport in cells remain unclear. The goal of this work is to use stochastic simulations of bidirectional transport to determine the motor properties that most strongly determine overall cargo velocity and directionality. Simulations were carried out based on published optical tweezer characterization of kinesin‐1 and kinesin‐2, and for available data for cytoplasmic dynein and the dynein‐dynactin‐BicD2 (DDB) complex. By varying dynein parameters and analyzing cargo trajectories, we find that net cargo transport is predicted to depend minimally on the dynein stall force, but strongly on dynein load‐dependent detachment kinetics. In simulations, dynein is dominated by kinesin‐1, but DDB and kinesin‐1 are evenly matched, recapitulating recent experimental work. Kinesin‐2 competes less well against dynein and DDB, and overall, load‐dependent motor detachment is the property that most determines a motor's ability to compete in bidirectional transport. It follows that the most effective intracellular regulators of bidirectional transport are predicted to be those that alter motor detachment kinetics rather than motor velocity or stall force.      

The “8-kD” Cytoplasmic Dynein Light Chain Is Required for Nuclear Migration and for Dynein Heavy Chain Localization in Aspergillus nidulans

The heavy chain of cytoplasmic dynein is required for nuclear migration in Aspergillus nidulans and other fungi. Here we report on a new gene required for nuclear migration, nudG, which encodes a homologue of the “8-kD” cytoplasmic dynein light chain (CDLC). We demonstrate that the temperature sensitive nudG8 mutation inhibits nuclear migration and growth at restrictive temperature. This mutation also inhibits asexual and sexual sporulation, decreases the intracellular concentration of the nudG CDLC protein and causes the cytoplasmic dynein heavy chain to be absent from the mycelial tip, where it is normally located in wild-type mycelia. Coimmunoprecipitation experiments with antibodies against the cytoplasmic dynein heavy chain (CDHC) and the nudG CDLC demonstrated that some fraction of the cytoplasmic dynein light chain is in a protein complex with the CDHC. Sucrose gradient sedimentation analysis, however, showed that not all of the NUDG protein is complexed with the heavy chain. A double mutant carrying a cytoplasmic dynein heavy chain deletion plus a temperature-sensitive nudG mutation grew no more slowly at restrictive temperature than a strain with only the CDHC deletion. This result demonstrates that the effect of the nudG mutation on nuclear migration and growth is mediated through an interaction with the CDHC rather than with some other molecule (e.g., myosin-V) with which the 8-kD CDLC might theoretically interact.     

Cytoplasmic dynein and microtubule transport in the axon: The action connection

The neuron uses two families of microtubule-based motors for fast axonal transport, kinesin, and cytoplasmic dynein. Cytoplasmic dynein moves membranous organelles from the distal regions of the axon to the cell body. Because dynein is synthesized in the cell body, it must first be delivered to the axon tip. It has recently been shown that cytoplasmic dynein is moved from the cell body along the axon by two different mechanisms. A small amount is associated with fast anterograde transport, the membranous organelles moved by kinesin. Most of the dynein is transported in slow component b, the actin-based transport compartment. Dynactin, a protein complex that binds dynein, is also transported in slow component b. The dynein in slow component b binds to microtubules in an ATP-dependent manner in vitro, suggesting that this dynein is enzymatically active. The finding that functionally active dynein, and dynactin, are associated with the actin-based transport compartment suggests a mechanism whereby dynein anchored to the actin cytoskeleton via dynactin provides the motive force for microtubule movement in the axon.     

Indole-3-acetic acid transport in apical dominance: a quantitative approach. Influence of endogenous and exogenous IAA apical source on inhibitory power of IAA transport

The relationship between the amount of indole-3-acetic acid transported (IAA transport) through the second node of 7-day-old pea seedlings and the degree of inhibition of axillary bud outgrowth at the same node was studied. For both the endogenous apical IAA source (leaves of apical bud) and the exogenous one (lanolin paste containing 0.25–1.0 mg mL^–1 IAA) the slope of linear dependence between inhibition and IAA transport was similar. However, the same IAA transport induced different inhibitions, which were higher for the endogenous source. Moreover, the apical bud induced higher inhibition at the same level of IAA transport when the 4th leaf was present than when it was absent. Apparently, the source of IAA also may regulate the inhibitory power of IAA transported from it. IAA transport appears to consists of active and slightly active one moving along different pathways.Abbreviations a and b coefficients of linear regression of the type y = a+bx; - confidence level of t-test - ELISA enzyme linked immunosorbent assay - GR_1,2 ^e/d growth rate of the lateral bud of experimental/decapitated (control) pea plants at the first and second days after treatment or decapitation - I degree of inhibition of lateral bud outgrowth - IAA indole-3-acetic acid - L_1,2,3 the lengths of lateral bud at 1, 2 or 3rd day after treatment or decapitation of pea plants - n data number - r correlation coefficient - T amount of IAA transported through the second node of pea plant for 3 hours - TIBA 2, 3, 5-triiodobenzoic acid - t-test statistical test used here to compare slopes of linear regressions (y = a+bx) calculated as % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x% fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaGaaeiDaiaabc% cacaqG9aGaaeiiaiaadkgadaWgaaWcbaGaaGymaaqabaGccaqGGaGa% aeylaiaabccacaWGIbWaaSbaaSqaaiaaikdaaeqaaOGaaeiiaiaab+% cacaqGGaWaaOaaaeaacaqGBbaaleqaaOGaaeikaiaabohacaqGLbGa% aeiiaiaadkgadaWgaaWcbaGaaGymaaqabaGccaqGPaWaaWbaaSqabe% aacaqGYaaaaOGaaeiiaiaabUcacaqGGaGaaeikaiaabohacaqGLbGa% aeiiaiaadkgadaWgaaWcbaGaaGOmaaqabaGccaqGPaWaaWbaaSqabe% aacaqGYaaaaOGaaeyxaiaab6caaaa!524A!\[{\text{t = }}b_1 {\text{ - }}b_2 {\text{ / }}\sqrt {\text{[}} {\text{(se }}b_1 {\text{)}}^{\text{2}} {\text{ + (se }}b_2 {\text{)}}^{\text{2}} {\text{]}}{\text{.}}\]     

Antagonistic forces generated by cytoplasmic dynein and myosin-II during growth cone turning and axonal retraction

Cytoplasmic dynein transports short microtubules down the axon in part by pushing against the actin cytoskeleton. Recent studies have suggested that comparable dynein-driven forces may impinge upon the longer microtubules within the axon. Here, we examined a potential role for these forces on axonal retraction and growth cone turning in neurons partially depleted of dynein heavy chain (DHC) by small interfering RNA. While DHC-depleted axons grew at normal rates, they retracted far more robustly in response to donors of nitric oxide than control axons, and their growth cones failed to efficiently turn in response to substrate borders. Live cell imaging of dynamic microtubule tips showed that microtubules in DHC-depleted growth cones were largely confined to the central zone, with very few extending into filopodia. Even under conditions of suppressed microtubule dynamics, DHC depletion impaired the capacity of microtubules to advance into the peripheral zone of the growth cone, indicating a direct role for dynein-driven forces on the distribution of the microtubules. These effects were all reversed by inhibition of myosin-II forces, which are known to underlie the retrograde flow of actin in the growth cone and the contractility of the cortical actin during axonal retraction. Our results are consistent with a model whereby dynein-driven forces enable microtubules to overcome myosin-II-driven forces, both in the axonal shaft and within the growth cone. These dynein-driven forces oppose the tendency of the axon to retract and permit microtubules to advance into the peripheral zone of the growth cone so that they can invade filopodia.     

CDK5‐dependent activation of dynein in the axon initial segment regulates polarized cargo transport in neurons

The unique polarization of neurons depends on selective sorting of axonal and somatodendritic cargos to their correct compartments. Axodendritic sorting and filtering occurs within the axon initial segment (AIS). However, the underlying molecular mechanisms responsible for this filter are not well understood. Here, we show that local activation of the neuronal‐specific kinase cyclin‐dependent kinase 5 (CDK5) is required to maintain AIS integrity, as depletion or inhibition of CDK5 induces disordered microtubule polarity and loss of AIS cytoskeletal structure. Furthermore, CDK5‐dependent phosphorylation of the dynein regulator Ndel1 is required for proper re‐routing of mislocalized somatodendritic cargo out of the AIS; inhibition of this pathway induces profound mis‐sorting defects. While inhibition of the CDK5‐Ndel1‐Lis1‐dynein pathway alters both axonal microtubule polarity and axodendritic sorting, we found that these defects occur on distinct timescales; brief inhibition of dynein disrupts axonal cargo sorting before loss of microtubule polarity becomes evident. Together, these studies identify CDK5 as a master upstream regulator of trafficking in vertebrate neurons, required for both AIS microtubule organization and polarized dynein‐dependent sorting of axodendritic cargos, and support an ongoing and essential role for dynein at the AIS.      

mNUDC is required for plus‐end‐directed transport of cytoplasmic dynein and dynactins by kinesin‐1

Lissencephaly is a devastating neurological disorder caused by defective neuronal migration. The LIS1 (or PAFAH1B1) gene was identified as the gene mutated in lissencephaly patients, and was found to regulate cytoplasmic dynein function and localization. In particular, LIS1 is essential for anterograde transport of cytoplasmic dynein as a part of the cytoplasmic dynein–LIS1–microtubule complex in a kinesin‐1‐dependent manner. However, the underlying mechanism by which a cytoplasmic dynein–LIS1–microtubule complex binds kinesin‐1 is unknown. Here, we report that mNUDC (mammalian NUDC) interacts with kinesin‐1 and is required for the anterograde transport of a cytoplasmic dynein complex by kinesin‐1. mNUDC is also required for anterograde transport of a dynactin‐containing complex. Inhibition of mNUDC severely suppressed anterograde transport of distinct cytoplasmic dynein and dynactin complexes, whereas motility of kinesin‐1 remained intact. Reconstruction experiments clearly demonstrated that mNUDC mediates the interaction of the dynein or dynactin complex with kinesin‐1 and supports their transport by kinesin‐1. Our findings have uncovered an essential role of mNUDC for anterograde transport of dynein and dynactin by kinesin‐1.     

Heteronuclear NMR identifies a nascent helix in intrinsically disordered dynein intermediate chain: implications for folding and dimerization

The intermediate chain of dynein forms a tight subcomplex with dimeric light chains LC8 and Tctex-1, and together they constitute the cargo attachment complex. There is considerable interest in identifying the role of these light chains in the assembly of the two copies of the intermediate chain. The N-terminal domain of the intermediate chain, IC1-289, contains the binding sites for the light chains, and is a highly disordered monomer but gains helical structure upon binding to light chains LC8 and Tctex-1. To provide insights into the structural and dynamic changes that occur in the intermediate chain upon light chains binding, we have used NMR spectroscopy to compare the properties of two distinct sub-domains of IC1-289: IC84-143 which is the light chains binding domain, and IC198-237, which contains a predicted coiled coil necessary for the increase in ordered structure upon light chain binding. Neither construct has stable secondary structure when probed by circular dichroism and amide chemical shift dispersion. Specific residues of IC84-143 involved in binding to the light chains were identified by their increase in resonance line broadening and the corresponding large intensity reduction in 1H-15N HSQC spectra. Interestingly, IC84-143 shows no sign of structure formation after binding to either LC8 or Tctex-1 or to both. IC198-237, on the other hand, contains a population of a nascent helix at low temperature as identified by heteronuclear NMR relaxation measurements, secondary chemical shifts, and sequential amide-amide connectivities. These data are consistent with a model for light chain binding coupled to intermediate chain dimerization through forming a coiled coil distant from the binding site.     

Tau directs intracellular trafficking by regulating the forces exerted by kinesin and dynein teams

Organelles, proteins, and mRNA are transported bidirectionally along microtubules by plus‐end directed kinesin and minus‐end directed dynein motors. Microtubules are decorated by microtubule‐associated proteins (MAPs) that organize the cytoskeleton, regulate microtubule dynamics and modulate the interaction between motor proteins and microtubules to direct intracellular transport. Tau is a neuronal MAP that stabilizes axonal microtubules and crosslinks them into bundles. Dysregulation of tau leads to a range of neurodegenerative diseases known as tauopathies including Alzheimer's disease (AD). Tau reduces the processivity of kinesin and dynein by acting as an obstacle on the microtubule. Single‐molecule assays indicate that kinesin‐1 is more strongly inhibited than kinesin‐2 or dynein, suggesting tau might act to spatially modulate the activity of specific motors. To investigate the role of tau in regulating bidirectional transport, we isolated phagosomes driven by kinesin‐1, kinesin‐2, and dynein and reconstituted their motility along microtubules. We find that tau biases bidirectional motility towards the microtubule minus‐end in a dose‐dependent manner. Optical trapping measurements show that tau increases the magnitude and frequency of forces exerted by dynein through inhibiting opposing kinesin motors. Mathematical modeling indicates that tau controls the directional bias of intracellular cargoes through differentially tuning the processivity of kinesin‐1, kinesin‐2, and dynein. Taken together, these results demonstrate that tau modulates motility in a motor‐specific manner to direct intracellular transport, and suggests that dysregulation of tau might contribute to neurodegeneration by disrupting the balance of plus‐ and minus‐end directed transport.      

Purification and crystal structure of human ODA16: Implications for ciliary import of outer dynein arms by the intraflagellar transport machinery

Motile cilia protrude from cell surfaces and are necessary to create movement of cells and fluids in the body. At the molecular level, cilia contain several dynein molecular motor complexes including outer dynein arms (ODAs) that are attached periodically to the ciliary axoneme, where they hydrolyse ATP to create the force required for bending and motility of the cilium. ODAs are preassembled in the cytoplasm and subsequently trafficked into the cilium by the intraflagellar transport (IFT) system. In the case of the green alga Chlamydomonas reinhardtii, the adaptor protein ODA16 binds to ODAs and directly to the IFT complex component IFT46 to facilitate the ciliary import of ODAs. Here, we purified recombinant human IFT46 and ODA16, determined the high‐resolution crystal structure of the ODA16 protein, and carried out direct interaction studies of IFT46 and ODA16. The human ODA16 C‐terminal 320 residues adopt the fold of an eight‐bladed β‐propeller with high overall structural similarity to the Chlamydomonas ODA16. However, the small 80 residue N‐terminal domain, which in Chlamydomonas ODA16 is located on top of the β‐propeller and is required to form the binding cleft for IFT46, has no visible electron density in case of the human ODA16 structure. Furthermore, size exclusion chromatography and pull‐down experiments failed to detect a direct interaction between human ODA16 and IFT46. These data suggest that additional factors may be required for the ciliary import of ODAs in human cells with motile cilia.     

Solution structure of isoform 1 of Roadblock/LC7, a light chain in the dynein complex

Roadblock/LC7 is a member of a class of dynein light chains involved in regulating the function of the dynein complex. We have determined the three-dimensional structure of isoform 1 of the mouse Roadblock/LC7 cytoplasmic dynein light chain (robl1_mouse) by NMR spectroscopy. In contrast to a previously reported NMR structure of the human homolog with 96% sequence identity (PDB 1TGQ), which showed the protein as a monomer, our results indicate clearly that robl1 exists as a symmetric homodimer. The two beta3-strands pair with each other and form a continuous ten-stranded beta-sheet. The 25-residue alpha2-helix from one subunit packs antiparallel to that of the other subunit on the face of the beta-sheet. Zipper-like hydrophobic contacts between the two helices serve to stabilize the dimer. Through an NMR titration experiment, we localized the site on robl1_mouse that interacts with the 40 residue peptide spanning residues 243 through 282 of IC74-1_rat. These results provide physical evidence for a symmetrical interaction between dimeric robl1 and the two molecules of IC74-1 in the dynein complex.