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We examined the distribution of the dynein-associated protein NudE in Drosophila larval brain neuroblasts and spermatocytes, and analyzed the phenotypic consequences of a nudE null mutation. NudE can associate with kinetochores, spindles and the nuclear envelope. In nudE mutant brain mitotic cells, centrosomes are often detached from the poles. Mor...
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... of spindles with antibody against tubulin revealed that nudE mutations also cause defects in mitotic spindle morphology and chromosome congression. In contrast to the wild type, where essentially all spindles appear to be normal, only a small minority of nudE 39A cells had straight, bipolar spindles ( Fig. 3; Table 2). The most frequent defects were broad, unfocused spindle poles, and, as seen by staining for the centrosomal component DSpd-2 ( Giansanti et al., 2008), the failure of centrosomes to remain attached to the spindles. ...
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... these differences in the normal mechanisms of centrosome separation, centrosomes in nudE mutant neuroblasts and spermatocytes are both detached from the spindle poles (Figs 3 and 4; Table 2). Centrosome detachment from the spindle poles has been previously observed in neuroblasts from lis1, glued and dynein heavy chain (dhc64) mutants (Robinson et al., 1999;Siller et al., 2005;Wojcik et al., 2001), as well as in neuroblasts defective for Asp ( Wakefield et al., 2001). ...
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... Nde1/Ndel1 participate in many, if not all, dynein-dependent processes and have overlapping functions (Bolhy et al., 2011;Lam et al., 2010;McKinley and Cheeseman, 2017;Monda and Cheeseman, 2018), which also appears to be the case at kinetochores. Nde1/Ndel1 localize to kinetochores independently of RZZ-Spindly (Chan et al., 2009;Liang et al., 2007;Simões et al., 2018;Stehman et al., 2007;Wainman et al., 2009;Wynne and Vallee, 2018), and both paralogs have been implicated in kinetochore dynein recruitment. RNAi-mediated knockdown of Ndel1 in HEK293T cells , antibody injection (anti-Nde1/Ndel1) into LLC-PK1 cells (Stehman et al., 2007) and a C. elegans null-mutant of the Nde1/Ndel1 homolog nud-2 (Simões et al., 2018) all reduce kinetochore levels of dynein, dynactin and Lis1. ...
... Nde1/Ndel1 and Lis1 are also implicated in the stripping of corona components Mitevska et al., 2022;Siller et al., 2005;Yan et al., 2003;Yang et al., 2003), which could reflect their role in dynein recruitment. A null mutant of NudE in D. melanogaster, however, abolishes poleward streaming of Rod in neuroblasts without affecting kinetochore dynein levels (Wainman et al., 2009), suggesting that NudE plays a more direct role as a positive regulator of stripping. Interestingly, a human Lis1 mutant with reduced (A) Autoinhibited Spindly adopts a closed conformation that involves an interaction between a segment of coiled-coil 2 (residues 276-306) and the CC1 box. ...
The microtubule minus-end-directed motility of cytoplasmic dynein 1 (dynein), arguably the most complex and versatile cytoskeletal motor, is harnessed for diverse functions, such as long-range organelle transport in neuronal axons and spindle assembly in dividing cells. The versatility of dynein raises a number of intriguing questions, including how is dynein recruited to its diverse cargo, how is recruitment coupled to activation of the motor, how is motility regulated to meet different requirements for force production and how does dynein coordinate its activity with that of other microtubule-associated proteins (MAPs) present on the same cargo. Here, these questions will be discussed in the context of dynein at the kinetochore, the supramolecular protein structure that connects segregating chromosomes to spindle microtubules in dividing cells. As the first kinetochore-localized MAP described, dynein has intrigued cell biologists for more than three decades. The first part of this Review summarizes current knowledge about how kinetochore dynein contributes to efficient and accurate spindle assembly, and the second part describes the underlying molecular mechanisms and highlights emerging commonalities with dynein regulation at other subcellular sites.
... Comparing the 3 ′ ends of mRNAs expressed in testes from bam −/− ;hs-Bam flies before heat shock versus 48 or 72 h PHS revealed a set of ∼500 genes that expressed mRNA isoforms with long 3 ′ UTRs at the 0-h time point, when the testes are filled with proliferating spermatogonia, but novel mRNA isoforms with shorter 3 ′ UTRs at 48 or 72 h PHS, when the testes have many early or mid-stage spermatocytes in addition to spermatogonia. For example, plotting the 3 ′ -seq reads on genomic regions starting just before the stop codon and extending downstream for nudE (ortholog of mammalian NDEL1 [NudE neurodevelopment protein 1-like 1]) (Sasaki et al. 2000;Wainman et al. 2009) and discs overgrown (dco; ortholog of mammalian CKIε) (Fig. 1B,C;Jursnich et al. 1990;Kloss et al. 1998) revealed that the 3 ′ end cut site used in testes filled with spermatogonia mapped 615 nt (nudE) or 1557 nt (dco) downstream from the stop codon. In contrast, 3 ′ -seq from testes 72 h PHS featured a new 3 ′ end cut and polyadenylation site much closer to the stop codon (121 nt for nudE; 122 nt for dco). ...
... Strikingly, for more than half (200 out of 384), the isoform with the short 3 ′ UTR expressed from the same gene was almost exclusively present in the free, 40S, and/or 60S fractions in testis extracts from 48 h PHS, indicating translational repression (Fig. 4F). Immunofluorescence staining of testes with available antibodies against the protein product of one of these genes, NudE (Wainman et al. 2009), revealed that the protein is strongly expressed in spermatogonia but is abruptly down-regulated in the young spermatocytes differentiating in testes 48 h PHS (Fig. 4G-I), as predicted from the migration behavior of the mRNA isoforms upon polysome fractionation (Fig. 4E,F; Supplemental Fig. S6A). Levels of immunofluorescence signal for NudE protein remained low in mid-stage spermatocytes present at 72 h PHS and the maturing spermatocytes present at 96 h PHS (Fig. 4J,K, dotted brackets), although high levels of immunofluorescence were detected in the bam −/− spermatogonia accumulating at the apical tip of the testes (Fig. 4J,K, solid brackets). ...
... The sources and dilutions of primary antibodies used were as follows: Vasa (goat; 1:100; Santa Cruz Biotechnology dc-13), Lola-F (mouse; 1:100; DSHB 1F1-1D5), Kumgang (rabbit; 1;400) (Kim et al. 2017), NudE (rabbit; 1:200) (Wainman et al. 2009), Orb (mouse; orb 4H8 and orb 6H4; 1:30; DSHB), and Numb (guinea pig; 1:500) (O'Connor-Giles and Skeath 2003). ...
Alternative polyadenylation (APA) generates transcript isoforms that differ in the position of the 3′ cleavage site, resulting in the production of mRNA isoforms with different length 3′ UTRs. Although widespread, the role of APA in the biology of cells, tissues, and organisms has been controversial. We identified >500 Drosophila genes that express mRNA isoforms with a long 3′ UTR in proliferating spermatogonia but a short 3′ UTR in differentiating spermatocytes due to APA. We show that the stage-specific choice of the 3′ end cleavage site can be regulated by the arrangement of a canonical polyadenylation signal (PAS) near the distal cleavage site but a variant or no recognizable PAS near the proximal cleavage site. The emergence of transcripts with shorter 3′ UTRs in differentiating cells correlated with changes in expression of the encoded proteins, either from off in spermatogonia to on in spermatocytes or vice versa. Polysome gradient fractionation revealed >250 genes where the long 3′ UTR versus short 3′ UTR mRNA isoforms migrated differently, consistent with dramatic stage-specific changes in translation state. Thus, the developmentally regulated choice of an alternative site at which to make the 3′ end cut that terminates nascent transcripts can profoundly affect the suite of proteins expressed as cells advance through sequential steps in a differentiation lineage.
... Comparing the 3 ′ ends of mRNAs expressed in testes from bam −/− ;hs-Bam flies before heat shock versus 48 or 72 h PHS revealed a set of ∼500 genes that expressed mRNA isoforms with long 3 ′ UTRs at the 0-h time point, when the testes are filled with proliferating spermatogonia, but novel mRNA isoforms with shorter 3 ′ UTRs at 48 or 72 h PHS, when the testes have many early or mid-stage spermatocytes in addition to spermatogonia. For example, plotting the 3 ′ -seq reads on genomic regions starting just before the stop codon and extending downstream for nudE (ortholog of mammalian NDEL1 [NudE neurodevelopment protein 1-like 1]) (Sasaki et al. 2000;Wainman et al. 2009) and discs overgrown (dco; ortholog of mammalian CKIε) (Fig. 1B,C;Jursnich et al. 1990;Kloss et al. 1998) revealed that the 3 ′ end cut site used in testes filled with spermatogonia mapped 615 nt (nudE) or 1557 nt (dco) downstream from the stop codon. In contrast, 3 ′ -seq from testes 72 h PHS featured a new 3 ′ end cut and polyadenylation site much closer to the stop codon (121 nt for nudE; 122 nt for dco). ...
... Strikingly, for more than half (200 out of 384), the isoform with the short 3 ′ UTR expressed from the same gene was almost exclusively present in the free, 40S, and/or 60S fractions in testis extracts from 48 h PHS, indicating translational repression (Fig. 4F). Immunofluorescence staining of testes with available antibodies against the protein product of one of these genes, NudE (Wainman et al. 2009), revealed that the protein is strongly expressed in spermatogonia but is abruptly down-regulated in the young spermatocytes differentiating in testes 48 h PHS (Fig. 4G-I), as predicted from the migration behavior of the mRNA isoforms upon polysome fractionation (Fig. 4E,F; Supplemental Fig. S6A). Levels of immunofluorescence signal for NudE protein remained low in mid-stage spermatocytes present at 72 h PHS and the maturing spermatocytes present at 96 h PHS (Fig. 4J,K, dotted brackets), although high levels of immunofluorescence were detected in the bam −/− spermatogonia accumulating at the apical tip of the testes (Fig. 4J,K, solid brackets). ...
... The sources and dilutions of primary antibodies used were as follows: Vasa (goat; 1:100; Santa Cruz Biotechnology dc-13), Lola-F (mouse; 1:100; DSHB 1F1-1D5), Kumgang (rabbit; 1;400) (Kim et al. 2017), NudE (rabbit; 1:200) (Wainman et al. 2009), Orb (mouse; orb 4H8 and orb 6H4; 1:30; DSHB), and Numb (guinea pig; 1:500) (O'Connor-Giles and Skeath 2003). ...
Alternative polyadenylation (APA) generates transcript isoforms that differ in the position of the 3’ cleavage site, resulting in the production of mRNA isoforms with different length 3’UTRs. Although widespread, the role of APA in the biology of cells, tissues and organisms has been controversial. We identified over 500 Drosophila genes that express mRNA isoforms with a long 3’UTR in proliferating spermatogonia but a short 3’UTR in differentiating spermatocytes due to APA. We show that the stage-specific choice of the 3’ end cleavage site can be regulated by the arrangement of a canonical polyadenylation signal (PAS) near the distal cleavage site but a variant or no recognizable PAS near the proximal cleavage site. The emergence of transcripts with shorter 3’UTRs in differentiating cells correlated with changes in expression of the encoded proteins, either from off in spermatogonia to on in spermatocytes or vice versa. Polysome gradient fractionation revealed over 250 genes where the long 3’UTR versus short 3’UTR mRNA isoforms migrated differently, consistent with dramatic stage-specific changes in translation state. Thus, the developmentally regulated choice of an alternative site at which to make the 3’end cut that terminates nascent transcripts can profoundly affect the suite of proteins expressed as cells advance through sequential steps in a differentiation lineage.
... There is evidence that Nde1/Ndel1 support nearly all of dynein's mitotic dynein functions. Both Nde1 and Ndel1 drive dynein localization to the nuclear envelope to promote nuclear envelope breakdown and positioning of centrosomes, ( Figure 3E), promote spindle formation and focusing ( Figure 3F), and facilitate dynein localization at the kinetochore in prometaphase ( Figure 3G) (Liang et al., 2007;Mori et al., 2007;Stehman et al., 2007;Vergnolle and Taylor, 2007;Hebbar et al., 2008;Wainman et al., 2009;Bolhy et al., 2011;Wang and Zheng, 2011;Żyłkiewicz et al., 2011;Monda and Cheeseman, 2018;Wynne and Vallee, 2018). Although Nde1 and Ndel1 seem capable of fulfilling similar roles during cell division, Nde1 drives dynein localization at kinetochores to a greater extent than Ndel1 (Vergnolle and Taylor, 2007). ...
Cytoplasmic dynein-1 (dynein) is the primary microtubule minus-end directed molecular motor in most eukaryotes. As such, dynein has a broad array of functions that range from driving retrograde-directed cargo trafficking to forming and focusing the mitotic spindle. Dynein does not function in isolation. Instead, a network of regulatory proteins mediate dynein’s interaction with cargo and modulate dynein’s ability to engage with and move on the microtubule track. A flurry of research over the past decade has revealed the function and mechanism of many of dynein’s regulators, including Lis1, dynactin, and a family of proteins called activating adaptors. However, the mechanistic details of two of dynein’s important binding partners, the paralogs Nde1 and Ndel1, have remained elusive. While genetic studies have firmly established Nde1/Ndel1 as players in the dynein transport pathway, the nature of how they regulate dynein activity is unknown. In this review, we will compare Ndel1 and Nde1 with a focus on discerning if the proteins are functionally redundant, outline the data that places Nde1/Ndel1 in the dynein transport pathway, and explore the literature supporting and opposing the predominant hypothesis about Nde1/Ndel1’s molecular effect on dynein activity.
... Thus, the roles of dynein-dynactin during spindle assembly in spermatocytes resemble those in the early embryo. In D. melanogaster, the only other animal in which the consequences of dynein inhibition have been characterized in spermatocytes, mutations in dynein regulators (Asunder, Lis1, NudE) and dynein itself (dynein light chain Tctex) prevent the initial attachment of centrosomes to the prophase nucleus, which results in spindle assembly without centrosomes (Anderson et al., 2009;Wainman et al., 2009;Sitaram et al., 2012). This defect makes it difficult to evaluate the role of dynein at later stages of division. ...
The microtubule motor cytoplasmic dynein 1 (dynein) and its essential activator dynactin have conserved roles in spindle assembly and positioning during female meiosis and mitosis, but their contribution to male meiosis remains poorly understood. Here, we characterize the G33S mutation in the C. elegans dynactin subunit DNC-1, which corresponds to G59S in human p150 Glued that causes motor neuron disease. In spermatocytes, dnc-1(G33S) delays spindle assembly and penetrantly inhibits anaphase spindle elongation in meiosis I, which prevents the segregation of homologous chromosomes. By contrast, chromosomes segregate without errors in the early dnc-1(G33S) embryo. Deletion of the DNC-1 N-terminus shows that defective meiosis in dnc-1(G33S) spermatocytes is not due to DNC-1's inability to interact with microtubules. Instead, our results suggest that the DNC-1(G33S) protein, which is aggregation-prone in vitro , is less stable in spermatocytes than the early embryo, resulting in different phenotypic severity in the two dividing tissues. Thus, the dnc-1(G33S) mutant reveals that dynein-dynactin drive meiotic chromosome segregation in spermatocytes and illustrates that the extent to which protein misfolding leads to loss of function can vary significantly between cell types.
... For example, the role of NDE1 in both nuclear migration and mitotic spindle regulation is well-conserved across eukaryotes (Morris et al., 1995;Alkuraya et al., 2011;Xiang, 2018). Meanwhile, evidence indicates that the kinetochore-binding function of NDE1 is conserved from Ecdysozoa to Primates (Wainman et al., 2009;Simões et al., 2017). Despite the conservation of NDE1 functions, its role at the organismal level differs across evolution. ...
An expanded cortex is a hallmark of human neurodevelopment and endows increased cognitive capabilities. Recent work has shown that the cell cycle-related gene NDE1 is essential for proper cortical development. Patients who have mutations in NDE1 exhibit congenital microcephaly as a primary phenotype. At the cellular level, NDE1 is essential for interkinetic nuclear migration and mitosis of radial glial cells, which translates to an indispensable role in neurodevelopment. The nuclear migration function of NDE1 is well conserved across Opisthokonta. In mammals, multiple isoforms containing alternate terminal exons, which influence the functionality of NDE1, have been reported. It has been noted that the pattern of terminal exon usage mirrors patterns of cortical complexity in mammals. To provide context to these findings, here, we provide a comprehensive review of the literature regarding NDE1, its molecular biology and physiological relevance at the cellular and organismal levels. In particular, we outline the potential roles of NDE1 in progenitor cell behavior and explore the spectrum of NDE1 pathogenic variants. Moreover, we assessed the evolutionary conservation of NDE1 and interrogated whether the usage of alternative terminal exons is characteristic of species with gyrencephalic cortices. We found that gyrencephalic species are more likely to express transcripts that use the human-associated terminal exon, whereas lissencephalic species tend to express transcripts that use the mouse-associated terminal exon. Among gyrencephalic species, the human-associated terminal exon was preferentially expressed by those with a high order of gyrification. These findings underscore phylogenetic relationships between the preferential usage of NDE1 terminal exon and high-order gyrification, which provide insight into cortical evolution underlying high-order brain functions.
... This dramatic redistribution of the ER from the cytoplasm to astral MTs around the centrosomes was clearly reflected by a four-fold increase in ER centrosome / ER cytoplasm at metaphase as compared to late interphase (6.45 ± 0.26 at metaphase vs. 1.60 ± 0.06 at interphase; Fig. 1c,d). In Dhc64C RNAi spermatocytes, the centrosomes separated but they failed to move from the cell cortex to the nuclear envelope, consistent with dynein's role in centrosome-nuclear envelope engagement [24][25][26] . Despite RNAi spermatocyte co-expressing GFP-tubulin (green) and RFP-KDEL (red) from late interphase through metaphase of the first meiotic division. ...
... Importantly, while the redistribution of ER to centrosomes during M-phase was not affected by Dhc64C RNAi, spindle architecture was greatly altered as expected with dynein suppression. Specifically, as Dhc64C RNAi cells progressed into a metaphase-like stage with congressed chromosomes and organized kinetochore spindle fibers, the centrosomes with their astral MTs were dissociated from the spindle poles, consistent with dynein suppression 26,27 ; in some cases the axis of the two centrosomes was completely perpendicular to the orientation of the spindle itself (Fig. 2b). Even with these dramatic defects in spindle architecture in Dhc64C RNAi spermatocytes, the ER remained specifically associated with the astral MTs around the centrosomes, as in control cells, suggesting that dynein is not required for M-phase redistribution of the ER to astral MTs around centrosomes. ...
... Further supporting this conclusion, the maximal ER centrosome /ER cytoplasm value attained in Dhc64C RNAi cells at metaphase was not significantly different from controls ( Fig. 2c,d). RNAi-mediated suppression of NudE, a dynein activator with multiple mitotic functions 26 , was used to further confirm the dynein-independence of ER redistribution to astral MTs in M-phase. To this end, the effects of NudE suppression were nearly identical to those of Dhc64C, on both ER redistribution and spindle architecture ( Supplementary Fig. S3). ...
In dividing animal cells the endoplasmic reticulum (ER) concentrates around the poles of the spindle apparatus by associating with astral microtubules (MTs), and this association is essential for proper ER partitioning to progeny cells. The mechanisms that associate the ER with astral MTs are unknown. Because astral MT minus-ends are anchored by centrosomes at spindle poles, we hypothesized that the MT minus-end motor dynein mediates ER concentration around spindle poles. Live in vivo imaging of Drosophila spermatocytes revealed that dynein is required for ER concentration around centrosomes during late interphase. In marked contrast, however, dynein suppression had no effect on ER association with astral MTs and concentration around spindle poles in early M-phase. In fact, there was a sudden onset of ER association with astral MTs in dynein RNAi cells, revealing activation of an M-phase specific mechanism of ER-MT association. ER redistribution to spindle poles also did not require non-claret disjunctional (ncd), the other known Drosophila MT minus-end motor, nor Klp61F, a MT plus-end motor that generates spindle poleward forces. Collectively, our results suggest that a novel, M-phase specific mechanism of ER-MT association that is independent of MT minus-end motors is required for proper ER partitioning in dividing cells.
... RNAi spermatocytes, the centrosomes separated but they failed to move from the cell cortex to the nuclear envelope, consistent with dynein's role in centrosome-nuclear envelope engagement (Robinson et al., 1999;Splinter et al., 2010;Wainman et al., 2009) This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. ...
... http://dx.doi.org/10.1101/574855 doi: bioRxiv preprint first posted online Mar. 12, 2019; consistent with dynein suppression (Morales-Mulia and Scholey, 2005;Wainman et al., 2009); in some cases the axis of the two centrosomes was completely perpendicular to the orientation of the spindle itself ( Figure 2b). Even with these dramatic defects in spindle architecture in Dhc64C ...
... Further supporting this conclusion, the maximal ERcentrosome / ERcytoplasm value attained in Dhc64C RNAi cells at metaphase was not significantly different from controls ( Figure 2c,d). RNAi-mediated suppression of NudE, a dynein activator with multiple mitotic functions (Wainman et al., 2009), was used to further confirm the dyneinindependence of ER redistribution to astral MTs in M-phase. To this end, the effects of NudE suppression were nearly identical to those of Dhc64C, on both ER redistribution and spindle architecture ( Supplementary Fig. S2). ...
In dividing animal cells the endoplasmic reticulum (ER) concentrates around the poles of the spindle apparatus by associating with astral microtubules (MTs), and this association is essential for proper ER partitioning to progeny cells. The mechanisms that associate the ER with astral MTs are unknown. Because astral MT minus-ends are anchored by centrosomes at spindle poles, we tested the hypothesis that the MT minus-end motor dynein mediates ER concentration around spindle poles. Live in vivo imaging of Drosophila spermatocytes undergoing the first meiotic division revealed that dynein is required for ER concentration around centrosomes during interphase. In marked contrast, however, dynein suppression had no effect on ER association with astral MTs and concentration around spindle poles in early M-phase. Importantly though, there was a sudden onset of ER-astral MT association in dynein RNAi cells, revealing activation of an M-phase specific mechanism. ER redistribution to spindle poles also did not require non-claret disjunctional (ncd), the other known Drosophila MT minus-end motor, nor Klp61F, a MT plus-end motor that generates spindle poleward forces. Collectively, our results suggest that a novel, M-phase specific mechanism of ER-MT association that is independent of MT minus-end motors is required for proper ER partitioning in dividing cells.
... The MI is approximately twofold higher in dind mutants than in wild type, indicating that dividing cells in mutant brains are delayed at some point in M phase (Table 1). The effect of dind mutations on larval neuroblast mitotic progression is strong but not as potent as certain other mutations that can result in a > 3-fold elevation in the MI (e.g., Gatti and Baker 1989;Mottier-Pavie et al. 2011;Wainman et al. 2009). The proportion of mitotic figures in dind mutant brains that were in anaphase was roughly fivefold lower than in wild-type controls ( Table 1). ...
... These Banastral^diamond-shaped spindles were often rotated with respect to the axis defined by the asters (Fig. 7b). A similar phenotype has been previously observed in primary spermatocytes of asp, lis1, and NudE mutants (Rebollo et al. 2004;Wainman et al. 2009) and has been attributed to the failure of the centrioles and their associated asters to detach from the plasma membrane and migrate towards the nuclear envelope as in wild type (Gunsalus et al. 1995;Rebollo et al. 2004). ...
Many genes are required for the assembly of the mitotic apparatus and for proper chromosome behavior during mitosis and meiosis. A fruitful approach to elucidate the mechanisms underlying cell division is the accurate phenotypic characterization of mutations in these genes. Here, we report the identification and characterization of diamond (dind), an essential Drosophila gene required both for mitosis of larval brain cells and for male meiosis. Larvae homozygous for any of the five EMS-induced mutations die in the third-instar stage and exhibit multiple mitotic defects. Mutant brain cells exhibit poorly condensed chromosomes and frequent chromosome breaks and rearrangements; they also show centriole fragmentation, disorganized mitotic spindles, defective chromosome segregation, endoreduplicated metaphases, and hyperploid and polyploid cells. Comparable phenotypes occur in mutant spermatogonia and spermatocytes. The dind gene encodes a non-conserved protein with no known functional motifs. Although the Dind protein exhibits a rather diffuse localization in both interphase and mitotic cells, fractionation experiments indicate that some Dind is tightly associated with the chromatin. Collectively, these results suggest that loss of Dind affects chromatin organization leading to defects in chromosome condensation and integrity, which in turn affect centriole stability and spindle assembly. However, our results do not exclude the possibility that Dind directly affects some behaviors of the spindle and centrosomes.
... Dynactin binds to microtubules and to dynein through its largest subunit, p150 Glued , and this interaction is required for recruitment of cargo to dynein (Karki and Holzbaur, 1999), for the processivity of dynein along microtubules (King and Schroer, 2000;, and for correct spindle formation and cell division (Godin et al., 2010). Another essential dynein interaction is with NudE and its homolog Nudel which regulates dynein recruitment to kinetochores and membranes (Lam et al., 2010), centrosome migration (Wainman et al., 2009), and mitotic spindle orientation (Lu and Prehoda, 2013). Since both dynactin p150 Glued and NudE bind dynein IC at a common site (Morgan et al., 2011;McKenney et al., 2011;Nyarko et al., 2012;Barbar, 2012), the selective binding of one protein versus another is tightly regulated, especially when both are present in the same cellular compartment. ...
Dynactin and NudE/Nudel are prominent regulators of cytoplasmic dynein motility and cargo-binding activities. Both interact with the intrinsically disordered N-terminal domain of dynein intermediate chain (IC), which also contains phosphorylation sites that apparently regulate these interactions. Nuclear magnetic resonance and isothermal calorimetry studies demonstrate that the Ser84 phosphorylation site identified in cells is in a disordered linker distant from the N-terminal helix that contains both the dynactin- and the Nudel-binding interfaces. Structural studies of a phosphomimetic Ser84Asp imply that phosphorylation stabilizes an electrostatic cluster that docks the disordered linker containing Ser84 against the N-terminal helix, resulting in a conformation that blocks access of IC to dynactin, but not to NudE/Nudel. Formation of this cluster is dependent on the length and sequence of the disordered linkers. This model explains the selective binding of mammalian IC to dynactin versus NudE/Nudel and why this selection is specific for IC-2C and not the IC-1A isoform.
