InteractiveFly: GeneBrief

Dynactin 2, p50 subunit:

Biological Overview | References

Dynamitin (Dmn) is a major component of dynactin, a multiprotein complex playing important roles in a variety of intracellular motile events. Previous work has found that Wolbachia bacterial infection resulted in a reduction of Dmn protein. Since Wolbachia may modify sperm in male hosts, it was speculated that Dmn may have a function in male fertility. This study used nosGal4 to drive Dmn knock down in testes of Drosophila melanogaster to investigate the functions of Dmn in spermatogenesis. Knockdown of Dmn in testes dramatically decreased male fertility, overexpression of Dmn in Wolbachia-infected males significantly rescued male fertility, indicating an important role of Dmn in inducing male fertility defects following Wolbachia infection. Some scattered immature sperm with late canoe-shaped head distributed in the end of Dmn knockdown testis and only about half mature sperm were observed in the Dmn knockdown testis relative to those in the control. Transmission electron microscopy (TEM) exhibited fused spermatids in cysts and abnormal mitochondrial derivatives. Immunofluorescence staining showed significantly less abundance of tubulin around the nucleus of spermatid and scattered F-actin cones to different extents in the individualization complex (IC) during spermiogenesis in Dmn knockdown testes, which may disrupt the nuclear condensation and sperm individualization. Since dynein-dynactin complex has been shown to mediate transport of many cellular components, including mRNAs and organelles, these results suggest that Dmn may play an important role in Drosophila spermiogenesis by affecting transport of many important cytoplasmic materials (Wu, 2016).

The Dynamitin (Dmn) gene encodes the p50 subunit of dynactin. Dmn protein is a core element of the dynactin complex structure: four Dmn subunits sit at the junction between dynactin's two major structural domains, the cargo-binding Arp1 filament and the p150Glued arm that supports interactions with motors and microtubules. Dynactin is a dynein accessory factor, which may enhance dynein's motor activity and play essential roles in regulating dynein's subcellular localization and interactions with other proteins (Wu, 2016).

The Drosophila melanogaster testis is an ideal model system for studying many biological processes, including the regulation of stem cells, meiosis, and sperm development. The stages of D. melanogaster spermatogenesis are well defined. Germline stem cells at the anterior tip give rise to spermatogonia, which undergo four synchronous mitotic divisions with incomplete cytokinesis to produce 16-cell cysts of primary spermatocytes. After two rounds of meiotic divisions, 64-cell cysts containing haploid spermatids are generated. During spermiogenesis from round spermatids to sperm, a multi-layered mitochondria aggregate and condense to form the nebenkern. Both nucleus and nebenkern associate with a centriole-derived basal body (BB), from which the flagellar axonemes are assembled and elongate. Finally the syncytial spermatids are individualized with the help of individualization complex (IC) composed of 64 actin cones, and the motile sperms with condensed needle-shape nuclei are released to the seminal vesicles (Wu, 2016).

In Drosophila, dynein regulates many aspects of development, including both male and female gametogenesis. Dynein-dynactin complex has been shown to play an essential role in spermatogenesis. It may anchor both centrosome and BB to the nucleus. In early G2 spermatocytes, dynein-dynactin diffuses throughout the cytoplasm. In late G2, however, dynein-dynactin accumulates at the nuclear periphery. Perinuclear dynein-dynactin may capture astral microtubules emanating from cortical centrosomes. Since dynein is a minus-end-directed microtubule motor, interaction between astral microtubules and anchored dynein-dynactin leads to the movement of centrosomes toward the nucleus, thus stable linkage between the nucleus and centrosomes is established, which is a prerequisite for fidelity of meiotic divisions. The coincident timing of ASUN's release to the cytoplasm and accumulation of dynein-dynactin on the nucleus imply that ASUN (asunder, previously known as Mat89Bb, essential regulator of spermatogenesis) may play a key role in mediating this cell cycle-dependent localization of dynein-dynactin. It has been found that mutation of sperm-associated antigen 4 (Spag4) resulted in sterility of male Drosophila. In the absence of Spag4, nuclei and centrioles or BBs dissociate from each other after meiosis. The dynein-dynactin failed to accumulate at the nuclear membrane, but instead, dynein-dynactin accumulated on the paired centrioles at the spermatocyte periphery. On round spermatids of Spag4 mutant, dynein-dynactin was not detected in the hemispherical cap as in wild types, but accumulated on the BB. This suggests that Spag4 may bring dynein-dynactin to the nuclear membrane, leading to subsequent downstream events for spermatogenesis. LIS1, another dynein accessory factor, directly binds several dynein and dynactin subunits through its C-terminal WD-repeat domain, and thus enhances dynein motor activity. Homozygous and hemizygous Lis-1 mutant males are uniformly infertile. In these sterile males, LIS-1 is required for recruiting dynein-dynactin to the nuclear surface and spindle poles of male germ cells. A pool of dynein anchored at the nuclear surface is thought to function in promoting stable interactions between the nucleus and centrosomes by mediating minus-end-directed movement of the nucleus along astral microtubules, thus LIS-1 may play an essential role in Drosophila spermatogenesis through dynein-dynactin's function (Wu, 2016).

By comparative proteomic analysis, previous work has found that Dmn protein was decreased in spermathecae and seminal receptacles of D. melanogaster females that mated with Wolbachia bacteria-infected males relative to those mated with uninfected males. As Wolbachia may modify sperm in male hosts and thus damage the male fertility, it is speculated that Dmn may have a function in male fertility. To investigate the role of Dmn in spermatogenesis, Dmn was knocked down in D. melanogaster testes by RNAi strategy. Knockdown of Dmn in male testes was found to result in dramatically decreased male fertility. By immunofluorescence technique and transmission electron microscopy (TEM), it was observed in the Dmn knockdown testes, the spermatid cell membrane frequently fused together, the immature sperm scattered around and bundles of individualization complexes (ICs) were disrupted. These phenotypes could be related to Lis-1 and Ddlc1 functions, since the expressions of Lis-1 and Ddlc1 were significantly reduced in the Dmn knockdown testes. These results suggest that Dmn may have an important function in spermiogenesis in D. melanogaster (Wu, 2016).

Wolbachia significantly modify host sperm and affect male fertility of their hosts. Previous work showed that Dmn protein was decreased in spermathecae and seminal receptacles of D. melanogaster females that mated with Wolbachia infected males relative to those mated with uninfected males. This study found that Dmn knockdown in male flies indeed severely impaired the male fertility. Wolbachia infection causes a more drastic downregulation in Dmn expression (14.2 fold) compared to nosGal4 driver (2.3 fold). Correspondingly, the male fertility is also different. Previous work have shown that Wolbachia-infected males gave only 8.78 ±1.03% of embryonic hatch rate (Zheng, 2011), while this study observed Dmn-hp knockdown males resulted in 28.97 ±4.14% of hatch rate. This suggests that the abundance of Dmn in testes is directly involved in male fertility. Furthermore, this study has shown that overexpression of Dmn in Wolbachia-infected male flies significantly rescue the fertility of both Dmn knockdown males and Wolbachia-infected males (Zheng, 2011), indicating that Dmn downregulation really plays an important role in inducing decreased male fertility in the presence of Wolbachia (Wu, 2016).

In Drosophila, spermiogenesis begins immediately after meiosis. During this stage the clonally related groups of 64 interconnected spermatids experience greatly morphological changes. One of the dramatic morphological changes is nuclear shaping and condensation. The shape of spermatid nucleus is changed from spherical to needle-like, and chromatin condenses as much as 200-fold. In the course of nuclear condensation, somatic constitutive histones are replaced by highly basic proteins such as protamine. The alteration of nuclear shape is driven by microtubules that emanate from the basal body (BB) and connect with the nuclear envelope. The perinuclear microtubules organize into parallel bundles, which are thought to provide support to the elongating nucleus. In the mean time, chromatin condensation occurs by switching from a histone-based chromatin structure in early round spermatid nuclei to a sperm-specific protein (SP, such as protamine)-based configuration in mature sperm nuclei. In Drosophila, the mammalian homologues of the protamine and transition protein have been identified as Protamine A (ProtA)/Protamine B (ProtB) and Transition protein like 94D (Tpl94D). This paper has shown that in the Dmn knockdown testes, some spermatid bundles were scattered before the nuclear shaping had completed. It has been reported that testis-specific β-tubulin has a pivotal role in the nuclear shaping in spermiogenesis. Cytoplasmic microtubules adjacent to the nuclei have been demonstrated to be important for nuclear shaping, which in turn may be required for the IC to assemble properly. This study did find that abundance of β-tubulin was significantly reduced around the nuclei in the Dmn knockdown spermatids than in the controls. This might result in the impairment of microtubule assembly. Hence less tubulin resulted from the Dmn knockdown might reduce the function of perinuclear microtubules, thus disrupt nuclear shaping. Some histone chaperones (such as Nap1) have been shown to play essential roles in nuclear shaping. Previous work also revealed that mutation of Hira, which encodes one of the histone H3.3 chaperones, significantly impaired male fertility in Drosophila. The role of Hira in spermiogenesis is not clear. However, HIRA has been demonstrated to have a critical function in sperm nuclear decondensation after fertilization by transforming a SP-based configuration to histone-based chromatin structure. Many cytoskeleton-related proteins were identified as histone chaperone partners. Therefore the transport property depending on Dmn may contribute to nuclear condensation and bundling through histone chaperone pathway and thus affect spermiogenesis. Consistently, during spermatogenesis of Wolbachia-infected male D. simulans, some nuclei were displaced from the apical cluster in some spermatid bundles. Thus, downregulation of Dmn in testes induced by Wolbachia infection, may be one reason that Wolbachia-infected males produce fewer progeny when crossed with Wolbachia-uninfected females (Wu, 2016).

The other dramatic change during Drosophila spermiogenesis is mitochondria. The mitochondria first aggregate at one side of the nucleus and then fuse and condense to form a nebenkern, which is connected with nucleus through BB. The mechanism by which mitochondria aggregate, fuse and condense to form the nebenkern near the nucleus is not well understood. This study found that there were multiple axoneme-nebenkern sets within a single cellular envelope in the Dmn knockdown testes. Two axoneme-nebenkern sets were also observed gathered together through two linked retracted minor mitochondrial derivatives. This suggests that interspermatid membrane deposition process is impaired in the Dmn mutant. Similar phenotypes were also observed in the rnRacGAP, Ddlc1 and parkin mutants. It was subsequently found that in the Dmn knockdown flies, the expression level of Ddlc1 was significantly decreased, indicating that Dmn could be involved in interspermatid membrane deposition, and knockdown of Dmn may result in the abnormal axoneme-mitochondrial assembly. Nevertheless, knockdown of Dmn did not affect the organization of axenome, implying that it is not likely to be involved in axonemal assembly. Furthermore, multiple mitochondrial derivatives were found associated with one axenome, which was previously reported in Lis-1 mutant. This study demonstrated that Lis-1 transcript was significantly reduced in the Dmn mutants. An earlier study showed that testis-specific knockout of Lis1 affected acrosomal vesicle fusion and disrupted spermiogenesis. The cytoplasmic Dynein-Dynactin complex is known to play a role in vesicular trafficking inside the cell in a variety of different cellular functions, including membrane fusion. In light of these evidences, it seems that Dynein-Dynactin-dependent vesicle transport would play an important role in the formation of cell envelope around each growing assembly of axoneme-mitochondria complex. Therefore, knockdown of Dmn may disrupt the Dynein-Dyanctin-dependent vesicle transport ability, thus damages the deposition of spermatid membrane (Wu, 2016).

At the end of spermatid morphogenesis, the mature sperm are invested with their own membranes in a process of individualization. Individualization requires formation of an individualization complex (IC). The IC is composed of 64 actin cones, one for each spermatid nucleus of the cyst. Actin filaments make a meshwork at the leading edge of the cones and are organized into parallel tight bundles at the rear of the cones. During individualization, the IC moves processively down the length of the cyst, extruding unneeded organelles and cytoplasm, and resolving intercellular bridges to encase each sperm cell in its own plasma membrane. This study showed that in the control testes, as individualization proceeded, the actin cone bundles of the IC moved synchronously away from the nuclei toward the caudal end of the cyst. However, in the Dmn knockdown testes, disorganized actin cones was observed, and some cones dissociated from the bundles and scattered around individually. It has been reported that several molecular pathways may contribute to the individualization process, including those involved in new plasma membrane deposition and microtubule cytoskeleton. Individualization during spermiogenesis is related to many processes that require deposition of new membrane, such as cytokinesis and spermatid elongation. These processes normally use vesicles to shuttle phospholipids from the Golg. Knockdown of Dmn may have resulted in the defect of vesicle trafficking ability and thus deposition of new membrane as discussed above. A number of vesicle trafficking factors are demonstrated to be required for individualization, including Shibire/Dynamin. Dynamin could play a role in actin deposition, perhaps with Ddlc1. Mutation of Ddlc1 disrupts synchronous movement of the actin cones and nuclear shaping and positioning during individualization. By qRT-PCR, significantly decrease in expression of Ddlc1 in the Dmn knockdown testes relative to the control fly testes. Additionally, overexpression of Ddlc1 significantly rescue the defects of male fertility caused by Dmn knockdown. Therefore, it is proposed that knockdown of Dmn dramatically impairs the transport ability of dynein-dynactin complex, thus damages the transportation of many important materials in cells. Recently, several evidences show that dynein-dynactin complex mediates transport of many cellular components, including mRNAs and organelles, and silencing Dmn causes significantly reduction in retrograde transportation of mitochondria. Given that during spermiogenesis, especially midway through nuclear shaping and spermatid elongation, a burst of post-meiotic transcription produces a set of mRNAs that are needed to be transported to the growing ends of the spermatid cysts, and numerous organelles, such as mitochondria and centrosomes, also need to be transported to special locations for remodeling and formation of sperm-specific organelles for fertilization and activation of development, knockdown of Dmn might affect male fertility by damaging transport ability for many important cellular contents and thus spermiogenesis (Wu, 2016).

Efficient endocytic uptake and maturation in Drosophila oocytes requires Dynamitin/p50

Dynactin is a multi-subunit complex that functions as a regulator of the Dynein motor. A central component of this complex is Dynamitin/p50 (Dmn). Dmn is required for endosome motility in mammalian cell lines. However, the extent to which Dmn participates in the sorting of cargo via the endosomal system is unknown. This study examined the endocytic role of Dmn using the Drosophila melanogaster oocyte as a model. Yolk proteins are internalized into the oocyte via clathrin-mediated endocytosis, trafficked through the endocytic pathway, and stored in condensed yolk granules. Oocytes that were depleted of Dmn contained fewer yolk granules than controls. In addition, these oocytes accumulated numerous endocytic intermediate structures. Particularly prominent were enlarged endosomes that were relatively devoid of yolk proteins. Ultrastructural and genetic analyses indicate that the endocytic intermediates are produced downstream of Rab5. Similar phenotypes were observed upon depleting Dynein heavy chain (Dhc) or Lis1. Dhc is the motor subunit of the Dynein complex and Lis1 is a regulator of Dynein activity. It is therefore proposed that Dmn performs its function in endocytosis via the Dynein motor. Consistent with a role for Dynein in endocytosis, the motor co-localized with the endocytic machinery at the oocyte cortex in an endocytosis-dependent manner. These results suggest a model whereby endocytic activity recruits Dynein to the oocyte cortex. The motor along with its regulators, Dynactin and Lis1, functions to ensure efficient endocytic uptake and maturation (Liu, 2015).

Dynactin/Dynein is required to maintain nuclear position within postmitotic Drosophila photoreceptor neurons

How a nucleus is positioned within a highly polarized postmitotic animal cell is not well understood. The Dynactin complex (a regulator of the microtubule motor protein Dynein) has been shown to be required to maintain the position of the nucleus within post-mitotic Drosophila photoreceptor neurons. Multiple independent disruptions of Dynactin function cause a relocation of the photoreceptor nucleus toward the brain, and inhibiting Dynactin causes the photoreceptor to acquire a bipolar appearance with long leading and trailing processes. It has been found that while the minus-end directed motor Dynein cooperates with Dynactin in positioning the photoreceptor nucleus, the plus-end directed microtubule motor Kinesin acts antagonistically to Dynactin. These data suggest that the maintenance of photoreceptor nuclear position depends on a balance of plus-end and minus-end directed microtubule motor function (Whited, 2004).

The Dynactin complex is an assembly of 11 different subunits that functions as an activator of Dynein, serving as an adaptor for cargo and enhancing motor processivity. The Dynactin subunit Glued couples Dynactin to Dynein by binding to the Dynein intermediate chain (Dic, encoded by short wing). Overexpression of a truncated form of Glued that binds to Dic but cannot associate with the rest of the Dynactin complex acts as a powerful inhibitor of Dynein and Dynactin function. Overexpression of the Dynactin subunit Dynamitin disrupts Dynactin complex assembly and also inhibits Dynactin function. Biochemical studies have shown that the Dynactin complex also contains Capping Protein, a heterodimer composed of the Capping Protein alpha (Cpa) and Capping Protein beta (Cpb) subunits. Although best known for capping the barbed ends of filaments of actin, Capping Protein also associates with filaments of the actin-related Arp1 protein, which is a central element of the Dynactin complex (Whited, 2004 and references therein).

Patterning of the adult compound eye of Drosophila initiates during the third instar phase of larval life, and mutations in the Dynactin subunit Glued strongly disrupt eye development. Normally the nuclei of differentiating photoreceptors occupy apical regions of the eye disc. In animals heterozygous for the dominant-negative Glued allele Glued¹, many photoreceptor nuclei have been shown to accumulate within basal regions of the eye disc. The effect of Glued¹ on photoreceptor development was characterized using an antibody recognizing photoreceptor cell surfaces. In wild type, the region of the differentiating photoreceptor neuron containing the nucleus remained in the retina, while the photoreceptor axon extended through the optic stalk into the brain. However, in Glued¹ animals, while photoreceptors still extended axons into the brain, the region of the photoreceptor containing the nucleus often appeared to leave the retina and travel through the optic stalk into the brain. Staining of photoreceptor nuclei directly demonstrated the movement of photoreceptor nuclei out of the eye disc and into the optic stalk in Glued¹ mutants (Whited, 2004).

To further establish that Glued¹ defects reflected disruptions in Dynactin function, two other approaches were used to disrupt the Dynactin complex. Drosophila Dynamitin, which also inhibits Dynactin function in flies, was overexpressed in photoreceptor neurons. Loss-of-function mutations in the Dynactin subunit Cpb were examined by generating animals whose visual systems contained homozygous mutant clones of the cpb strong loss-of-function mutation cpb^M143. In these cpb^M143 mosaic animals, the nuclear regions of many photoreceptors were observed in the optic stalk and brain (Whited, 2004).

To confirm that the cpb^M143 mutant photoreceptor defect was due to a loss of cpb function, an additional strong loss-of-function cpb allele, cpb^F44, was isolated from an EMS mutagenesis and a chromosomal deficiency uncovering the cpb locus, Df(2L)E.2, was obtained. When animals contained homozygous mutant clones of cpb^F44 cells or homozygous mutant clones of Df(2L)E.2, a similar movement of photoreceptor nuclear regions toward the brain was observed. cpb/Df(2L)E.2 animals did not survive to third instar, preventing the classic genetic demonstration that these cpb alleles behaved as strong loss-of-function mutations. Fortunately, it was found that the [pYES-ß] genomic transgene, which contains the CPB coding region, was able to rescue the lethality of cpb/Df(2L)E.2 animals, but did not rescue the previously described cpb bristle defect. This suggested that [pYES-ß] was a partially functional rescue construct that could be used to examine the visual systems of otherwise cpb/Df(2L)E.2 animals. It was found that [pYES-ß];cpb^M143/Df(2L)E.2 animals display a photoreceptor defect similar to that of other cpb mutants, consistent with nuclear mispositioning resulting from the loss of cpb function. It was further confirmed that the defect was due to the loss of cpb function by successfully rescuing the cpb^M143/Df(2L)E.2 photoreceptor defects (as well as the cpb bristle defects) by expression of a wild-type Cpb cDNA under the control of a heterologous promoter. Staining of photoreceptor nuclei directly demonstrated the movement of photoreceptor nuclei out of the eye disc and into the optic stalk in cpb mutants (Whited, 2004).

The bifunctional nature of Cpb, which associates with filaments of actin as well as filaments of Arp1, means that loss of Cpb also increases filamentous actin levels (Hopmann, 2003). Nonetheless, previous studies have shown that increases in filamentous actin alone, such as those observed in hypomorphic cpb alleles or in actup mutants, do not cause photoreceptor nuclear mispositioning. Together with the Glued¹ and Dynamitin data, the cpb observations yield a consistent picture that alterations in Dynactin subunits cause mispositioning of photoreceptor cell bodies and nuclei, and indicate that Dynactin, and not just the Glued subunit, has an important role in photoreceptor development (Whited, 2004).

The mispositioning of photoreceptor nuclei in Dynactin mutants raised the question of whether these disruptions reflect altered positioning of the nucleus within the photoreceptor or simply migration of the entire photoreceptor. To address this question, single photoreceptors were labeled in wild type and in Glued¹ mutants. Wild-type photoreceptors exhibit a highly polarized morphology in which the region of the photoreceptor containing the nucleus lies in the apical region of the eye disc and an axon extends basally into the brain. Glued¹ mutant photoreceptors whose nuclei have entered the optic stalk had highly altered morphologies, with both leading and trailing processes extending from the regions of the cell where the misplaced nucleus was located. Leading and trailing processes of misplaced Glued¹ photoreceptors were quantified, considering only those with no other labeled cells or processes nearby. Of these 13 neurons, 12 had clearly detectable leading and trailing processes. The leading process (axon) extended into the target region and the trailing process extended back into the eye disc. These data demonstrate that inhibition of Dynactin function dramatically alters the position of the nucleus within the photoreceptor (Whited, 2004).

The Dynactin complex also controls the pattern of mitoses within the Drosophila retina. To determine whether nuclear mispositioning is a secondary consequence of the earlier mitotic requirement for Dynactin, the effects of specifically inhibiting the Dynactin complex in postmitotic photoreceptors was examined. Conditional inhibition of Dynactin function can be achieved through inducible expression of a truncated, dominant-negative form Glued (Glued^DN) that resembles the protein product of Glued¹. Glued^DN was expressed under the control of the postmitotic photoreceptor-specific Glass 38-1 promoter, which initiates expression in the photoreceptors only after their axons have entered the brain. Expression of Glued^DN under the control of Glass 38-1 caused photoreceptor nuclei to move into the optic stalk. Overexpression of Dynamitin under the control of Glass 38-1 caused similar photoreceptor nuclear positioning defects. These data demonstrate that Dynactin is required postmitotically in photoreceptors to maintain nuclear position and that the disruptions in nuclear positioning observed are not simply a secondary consequence of mitotic defects (Whited, 2004).

The displacement of photoreceptor nuclei from apical regions of the eye disc toward more basal regions could reflect an overall disruption in apical/basal polarity of the eye disc. The apical/basal polarity of developing photoreceptors was assessed by examining the distribution of the Drosophila ß-catenin Armadillo and the PDZ-domain-containing protein PATJ. Armadillo localizes to the zonula adherens separating the apical and basolateral membrane domains of developing photoreceptors, while PATJ localizes to the apical membrane domain. In wild-type eye discs, Armadillo is concentrated just beneath the apical tips of the developing photoreceptors. In Glued¹ animals Armadillo was still present near apical regions of the eye disc, even in areas completely devoid of apical photoreceptor nuclei. Thus, this marker of apical/basal polarity was retained even when photoreceptor nuclei moved basally. Similar results were obtained when Glued¹ mutants were visualized in cross-section using both Armadillo and PATJ. Apical localization of PATJ and Armadillo were observed in Glued¹ and the relative apical/basal ordering of these markers was maintained. These data suggest that the alterations in photoreceptor morphology are not caused by a loss of apical/basal polarity within the developing photoreceptors (Whited, 2004).

Dynactin has important functions in the organization of the microtubule cytoskeleton in many systems. The microtubule cytoskeleton of developing photoreceptors is highly polarized, with microtubule minus ends concentrated apical to the nucleus as detected using antisera recognizing gamma-tubulin. A similar apical focus is observed when using the fusion protein Nod:LacZ, which often co-localizes with microtubule minus ends. The relatively ubiquitous expression of gamma-tubulin in the retina complicated the analysis of gamma-tubulin localization when retinal patterning was disrupted. Therefore, the effect of Glued on factors associated with the microtubule cytoskeleton was examined by expressing Nod:LacZ specifically in postmitotic photoreceptors. In animals expressing Glued^DN in postmitotic photoreceptors as well as in Glued¹ mutants, Nod:LacZ was no longer exclusively concentrated in apical regions of photoreceptors, but rather spread into the photoreceptor axons. Thus, while the overall apical/basal polarity of the photoreceptors was not disrupted in Glued mutants, the spatial organization of the microtubule cytoskeleton-associated factor Nod:LacZ was affected (Whited, 2004).

Dynactin activates the microtubule motor Dynein, and strong loss-of-function mutations in dynein intermediate chain (dic) are dominant enhancers of the rough eye phenotype of Glued¹ mutants. Since Dynein and Dynactin may play multiple roles together during eye development, the effect of a reduction in dic gene dosage upon photoreceptor nuclear positioning was examined in Glued¹ animals. A twofold reduction in dic gene dosage caused a further decrease in the number of photoreceptor nuclei in apical regions of Glued¹ mutant eye discs. This did not reflect a simple reduction in the number of photoreceptors generated; large numbers of photoreceptor nuclei were crowded at the base of the eye disc and entered the optic stalk in both animals. Thus, a larger fraction of photoreceptor nuclei left apical positions when the level of dic gene activity was reduced, consistent with Dynein and Dynactin acting together in this process (Whited, 2004).

To identify additional factors that interact with Dynactin to control nuclear positioning, a genetic screen was performed to identify genes that dominantly enhanced or suppressed the Glued¹ external eye phenotype. From a collection of approximately 1800 stocks containing transposon-induced lethal mutations, several stocks were identified that had no dominant effect on eye development in a wild-type background, but were dominant enhancers or suppressors of Glued¹. Two dominant suppressors of Glued¹, khc^k13219 and khc^k13314, were alleles of kinesin heavy chain (khc), which encodes a subunit of the plus-end directed microtubule motor kinesin. The interaction with Glued¹ was further confirmed using the null allele khc⁸. Examination of developing eye discs demonstrated that a twofold reduction of khc gene dosage greatly increased the number of photoreceptor nuclei present in apical regions of Glued¹ mutant eye discs. This suggested that khc acts antagonistically to Glued in photoreceptor nuclear positioning (Whited, 2004).

To determine whether khc mutations interacted with Glued¹ in postmitotic photoreceptors, khc gene dosage was reduced in animals expressing dominant-negative Glued under the control of the postmitotic Glass_38-1 promoter. Wild-type animals (n >50 hemispheres) or animals containing the dominant-negative Glued transgene without the Glass 38-1 promoter never contained photoreceptor nuclei within their optic stalks. Glass_38-1:Glued^DN animals contained an average of 11±1 photoreceptor nuclei within the optic stalk. However, Glass_38-1:Glued^DN animals heterozygous for either khc^k13314 or khc⁸ showed a significant reduction in the number of photoreceptor nuclei in the optic stalk. Thus, a twofold reduction in khc gene dosage suppressed the effects of postmitotic expression of dominant-negative Glued, consistent with Glued and khc acting antagonistically within differentiated photoreceptors to regulate nuclear positioning (Whited, 2004).

The interaction between Glued and khc in other photoreceptors was studied by examining the Bolwig organ, a cluster of 12 photosensitive neurons that differentiate during embryonic development and extend axons into the brain. By second and third instar larval stages, Bolwig photoreceptor nuclei are located near the anterior tip of the larva and their axons extend over the eye/antennal disc into the brain, a distance of >200 µm. In wild-type second instar animals, photoreceptor neuron differentiation has not yet begun in the eye disc and no neuronal nuclei are present there. However, when Glued^DN was expressed in postmitotic Bolwig photoreceptors, their nuclei appeared on the surface of the eye/antennal disc. Thus, as in the photoreceptors of the adult eye, expression of Glued^DN in Bolwig photoreceptors caused their nuclei to be positioned closer to their axon termini; in many cases, the Bolwig nuclei were over 150 µm closer than normal to their axon terminals in the brain (Whited, 2004).

The interaction between Glued and khc in Bolwig photoreceptors was assessed by counting the number of Bolwig nuclei on the surface of the eye/antennal disc. While wild-type and UAS:Glued^DN animals had no neuronal nuclei in this region, Glass_38-1:Glued^DN animals contained 7±1. A reduction of khc gene dosage in Glass_38-1:Glued^DN; khc^k13314/+ and Glass_38-1:Glued^DN; khc⁸/+ animals significantly reduced this to 4±1 and 3±1, respectively. These data further support the functional antagonism of Glued and khc in photoreceptor nuclear positioning (Whited, 2004).

Drosophila wingless and pair-rule transcripts localize apically by Dynein-mediated transport of RNA particles

Asymmetric mRNA localization targets proteins to their cytoplasmic site of function. The mechanism of apical localization of wingless and pair-rule transcripts in the Drosophila blastoderm embryo has been elucidated by directly visualizing intermediates along the entire path of transcript movement. After release from their site of transcription, mRNAs diffuse within the nucleus and are exported to all parts of the cytoplasm, regardless of their cytoplasmic destinations. Endogenous and injected apical RNAs assemble selectively into cytoplasmic particles that are transported apically along microtubules. Cytoplasmic dynein is required for correct localization of endogenous transcripts and apical movement of injected RNA particles. It is proposed that dynein-dependent movement of RNA particles is a widely deployed mechanism for mRNA localization (Wilkie, 2001).

To study the mechanism of apical localization, whether actin and/or MTs are necessary for localization of injected mRNA was tested by preinjecting cytoskeletal inhibitors 10 min before injecting the RNA. It was found that preinjection of Cytochalasin B, at concentrations that disrupt the organization of actin filaments, has no affect on Runt mRNA localization. However, a similar disruption of nuclear position has been observed in the cortical cytoplasm. In contrast, preinjection of colcemid, which destabilizes blastoderm MTs, disrupts runt, wingless, and fushi tarazu RNA localization almost entirely. It is concluded that an intact MT cytoskeleton is required for apical localization of injected RNA and that actin does not play a major role in the process. However, some minor role for actin in apical localization of RNA cannot be excluded (Wilkie, 2001).

Whether the localization of injected RNA occurs by minus end directed MT-dependent motor movement was tested by preinjecting embryos with antibodies against Drosophila cytoplasmic dynein heavy chain (dhc). Two independently raised monoclonal antibodies against dhc are each sufficient to inhibit RUN, FTZ, and WG mRNA apical localization in most, or all, embryos. Either one, the anti-dynein antibody or the colcemid injections, is sufficient to cause apical RNA to partly diffuse away from the site of injection in a similar manner to embryos injected with HB RNA alone. Injected apical RNA does not diffuse in the absence of anti-dynein antibodies or Colcemid preinjections. These results suggest that apical RNA is tethered to MTs by dynein and that dynein is required for the transport of RNA particles (Wilkie, 2001).

To further test the involvement of cytoplasmic dynein in apical transcript localization, RNA was injected into mutant cytoplasmic dynein heavy chain (Dhc64C) embryos. A marked reduction was found in the speed of movement of injected apical targeted RNAs in dynein mutants. Cytoplasmic dynein is essential for many cellular processes, so strong mutations in Dhc64C are homozygous lethal in Drosophila and cannot be studied at the blastoderm stage. Instead hypomorphic allelic combinations of Dhc64C, which are viable in trans due to intragenic complementation, were used. In two different allelic combinations of Dhc64C, injected RNA particles move at speeds 60% to 70% slower than they do in wild-type. Staining Dhc64C mutant embryos with anti-tubulin antibodies showsthat MT distribution is indistinguishable from wild-type, indicating that the reduced speed of localization is not due indirectly to a disruption of the MTs. Instead, the reduction in speed is likely to show a direct requirement for dynein in particle transport (Wilkie, 2001).

To test whether cytoplasmic dynein is also required for apical localization of endogenous transcripts, the effects of Dhc64C hypomorphic mutants and anti-dhc antibodies on the apical localization of endogenous FTZ transcripts was tested by in situ hybridization. As expected, hypomorphic Dhc64C mutants show no detectable effects on FTZ apical mRNA localization since injected RNA localizes correctly, but more slowly. In contrast, injection of anti-dhc antibody disrupts endogenous FTZ localization, leading to unlocalized stripes of ftz mRNA 20–30 min after injection. Given that FTZ mRNA has a half-life of 6 min in the blastoderm, the FTZ transcripts observed are likely to have been synthesized after the injection. It is concluded that endogenous apical mRNA localization is also dynein dependent (Wilkie, 2001).

Dynactin is a protein complex that is involved in coordinating the activities of cytoplasmic dynein, and is thought to be required for most forms of dynein-based transport. To test whether dynactin is also required for apical RNA localization, a large excess of p50/dynamitin is preinjected into embryos 10 min before injecting apically targeted RNA. p50/dynamitin causes a significant reduction in the speed of RNA particle movement. p50/dynamitin is a subunit of dynactin whose overexpression is a widely used method of disrupting the dynactin complex and demonstrating conclusively dynein-dependent motility. Dynactin is required for some cargo binding and for dynein processivity. It is concluded that apical transcript localization in the blastoderm embryo occurs by cytoplasmic dynein- and dynactin-mediated transport along MTs toward their minus ends (Wilkie, 2001).

It is thought that export and localization of apical mRNA in the blastoderm embryo can be divided into six distinct steps. (1) During or after completion of transcription and processing, transcripts are assembled into particles, which contain various hnRNPs and export factors, some of which may form part of the cytoplasmic localization machinery. (2) mRNA particles diffuse freely after release from the site of transcription and processing until they reach nuclear pore complexes (NPCs) on the nuclear periphery. (3) mRNA particles are exported through NPCs in all parts of the nuclear envelope. (4) The composition of the particles probably changes during export from the nucleus and in the cytoplasm to recruit dynein, dynactin, and associated proteins. (5) Particles attach to MTs and are actively transported to the apical cytoplasm. (6) Particle movement arrests in the apical cytoplasm, where they may associate with other particles and become anchored (Wilkie, 2001).

The first three steps of apical localization are thought to be common to most mRNAs, because they are essential universal processes in eukaryotic cells. However, the last three steps of the localization pathway are likely to vary among different kinds of transcripts, since the key determinant in sorting different mRNAs to their correct cytoplasmic destinations is presumably RNP particle composition in the cytoplasm. It is possible that some components required for cytoplasmic sorting are preassembled in the nucleus, as suggested by studies showing that the localization of injected FTZ mRNA depends on preincubation with the hnRNPA1 protein Squid. Indeed, a requirement for hnRNPs has also been shown for GRK mRNA localization in the oocyte, for myelin basic protein mRNA in rat oligodendrocytes, and for Vg1 transcripts in Xenopus oocytes. However, the data in this study show that injected protein-free apical RNA assembles in the cytoplasm into particles that localize correctly, arguing that all the factors needed to assemble competent localization particles can also be recruited in the cytoplasm (Wilkie, 2001).

Functions of Dynamitin orthologs in other species

Dynactin is involved in Lewy body pathology

Dynactin forms a protein complex with dynein that retrogradely transports cargo along microtubules. Dysfunction of this dynein-dynactin complex causes several neurodegenerative diseases such as Perry syndrome, motor neuron diseases and progressive supranuclear palsy. A Recent study reported colocalization of phosphorylated alpha-synuclein (p-SNCA) and the largest subunit of dynactin (DCTN1) in Lewy body (LB)-like structures in Perry syndrome. Previous reports have not focused on the relationship between dynactin and synucleinopathies. Thus, autopsied human brains were examined from patients with Parkinson's disease, dementia with LBs, and multiple system atrophy using immunohistochemistry for p-SNCA, DCTN1, dynactin 2 (DCTN2, dynamitin) and dynein cytoplasmic 1 intermediate chain 1 (DYNC1I1). Microtubule affinity-regulating kinases (MARKs), which phosphorylate microtubule-associated proteins and trigger microtubule disruption, were also examined. Both brainstem-type and cortical LBs were immunopositive for DCTN1, DCTN2, DYNC1I1 and p-MARK and their staining often overlapped with p-SNCA. Lewy neurites were also immunopositive for DCTN1, DCTN2 and DYNC1I1. However, p-SNCA-positive inclusions of multiple system atrophy, which included both glial and neuronal cytoplasmic inclusions, were immunonegative for DCTN1, DCTN2, DYNC1I1 and p-MARK. Thus, immunohistochemistry for dynein-dynactin complex molecules, especially DCTN1, can clearly distinguish LBs from neuronal cytoplasmic inclusions. These results suggest that dynactin is closely associated with LB pathology (Shen, 2018).

The dynactin complex maintains the integrity of metaphasic centrosomes to ensure transition to anaphase

The dynactin complex is required for activation of the dynein motor complex, which plays a critical role in various cell functions including mitosis. During metaphase, the dynein-dynactin complex removes spindle checkpoint proteins from kinetochores to facilitate the transition to anaphase. Three components (p150(Glued), dynamitin, and p24) compose a key portion of the dynactin complex, termed the projecting arm. To investigate the roles of the dynactin complex in mitosis, RNA interference was used to down-regulate p24 and p150(Glued) in human cells. In response to p24 down-regulation, cells were observed with delayed metaphase in which chromosomes frequently align abnormally to resemble a "figure eight," resulting in cell death. The figure eight chromosome alignment is attributed to impaired metaphasic centrosomes that lack spindle tension. Like p24, RNA interference of p150(Glued) also induces prometaphase and metaphase delays; however, most of these cells eventually enter anaphase and complete mitosis. These findings suggest that although both p24 and p150(Glued) components of the dynactin complex contribute to mitotic progression, p24 also appears to play a role in metaphase centrosome integrity, helping to ensure the transition to anaphase (Ozaki, 2011).

Evolution of the eukaryotic dynactin complex, the activator of cytoplasmic dynein

Dynactin is a large multisubunit protein complex that enhances the processivity of cytoplasmic dynein and acts as an adapter between dynein and the cargo. It is composed of eleven different polypeptides of which eight are unique to this complex, namely dynactin1 (p150(Glued)), dynactin2 (p50 or dynamitin), dynactin3 (p24), dynactin4 (p62), dynactin5 (p25), dynactin6 (p27), and the actin-related proteins Arp1 and Arp10 (Arp11). To reveal the evolution of dynactin across the eukaryotic tree the presence or absence of all dynactin subunits was determined in most of the available eukaryotic genome assemblies. Altogether, 3061 dynactin sequences from 478 organisms have been annotated. Phylogenetic trees of the various subunit sequences were used to reveal sub-family relationships and to reconstruct gene duplication events. Especially in the metazoan lineage, several of the dynactin subunits were duplicated independently in different branches. The largest subunit repertoire is found in vertebrates. Dynactin diversity in vertebrates is further increased by alternative splicing of several subunits. The most prominent example is the dynactin1 gene, which may code for up to 36 different isoforms due to three different transcription start sites and four exons that are spliced as differentially included exons. The dynactin complex is a very ancient complex that most likely included all subunits in the last common ancestor of extant eukaryotes. The absence of dynactin in certain species coincides with that of the cytoplasmic dynein heavy chain: Organisms that do not encode cytoplasmic dynein like plants and diplomonads also do not encode the unique dynactin subunits. The conserved core of dynactin consists of dynactin1, dynactin2, dynactin4, dynactin5, Arp1, and the heterodimeric actin capping protein. The evolution of the remaining subunits dynactin3, dynactin6, and Arp10 is characterized by many branch- and species-specific gene loss events (Hammesfahr, 2012).

Dynamitin affects cell-surface expression of voltage-gated sodium channel Nav1.5

The major cardiac voltage-gated sodium channel Nav1.5 associates with proteins that regulate its biosynthesis, localization, activity and degradation. Identification of partner proteins is crucial for a better understanding of the channel regulation. Using a yeast two-hybrid screen, dynamitin was idenified as a Nav1.5-interacting protein. Dynamitin is part of the microtubule-binding multiprotein complex dynactin. When overexpressed it is a potent inhibitor of dynein/kinesin-mediated transport along the microtubules by disrupting the dynactin complex and dissociating cargoes from microtubules. The use of deletion constructs showed that the C-terminal domain of dynamitin is essential for binding to the first intracellular interdomain of Nav1.5. Co-immunoprecipitation assays confirmed the association between Nav1.5 and dynamitin in mouse heart extracts. Immunostaining experiments showed that dynamitin and Nav1.5 co-localize at intercalated discs of mouse cardiomyocytes. The whole-cell patch-clamp technique was applied to test the functional link between Nav1.5 and dynamitin. Dynamitin overexpression in HEK-293 (human embryonic kidney 293) cells expressing Nav1.5 resulted in a decrease in sodium current density in the membrane with no modification of the channel-gating properties. Biotinylation experiments produced similar information with a reduction in Nav1.5 at the cell surface when dynactin-dependent transport was inhibited. The present study strongly suggests that dynamitin is involved in the regulation of Nav1.5 cell-surface density (Chatin, 2014).

Dynactin integrity depends upon direct binding of dynamitin to Arp1

Dynactin is a multiprotein complex that works with cytoplasmic dynein and other motors to support a wide range of cell functions. It serves as an adaptor that binds both dynein and cargoes and enhances single-motor processivity. The dynactin subunit dynamitin (also known as p50) is believed to be integral to dynactin structure because free dynamitin displaces the dynein-binding p150(Glued) subunit from the cargo-binding Arp1 filament. This study shows that the intrinsically disordered dynamitin N-terminus binds to Arp1 directly. When expressed in cells, dynamitin amino acids (AA) 1-87 causes complete release of endogenous dynamitin, p150, and p24 from dynactin, leaving behind Arp1 filaments carrying the remaining dynactin subunits (CapZ, p62, Arp11, p27, and p25). Tandem-affinity purification-tagged dynamitin AA 1-87 binds the Arp filament specifically, and binding studies with purified native Arp1 reveal that this fragment binds Arp1 directly. Neither CapZ nor the p27/p25 dimer contributes to interactions between dynamitin and the Arp filament. This work demonstrates for the first time that Arp1 can directly bind any protein besides another Arp and provides important new insight into the underpinnings of dynactin structure (Cheong, 2014).

Cytoplasmic dynein promotes HIV-1 uncoating

Retroviral capsid (CA) cores undergo uncoating during their retrograde transport (toward the nucleus), and/or after reaching the nuclear membrane. However, whether HIV-1 CA core uncoating is dependent upon its transport is not understood. There is some evidence that HIV-1 cores retrograde transport involves cytoplasmic dynein complexes translocating on microtubules. This study investigated the role of dynein-dependent transport in HIV-1 uncoating. To interfere with dynein function, dynein heavy chain (DHC) was depleted using RNA interference, and p50/dynamitin was overexpressed. In immunofluorescence microscopy experiments, DHC depletion caused an accumulation of CA foci in HIV-1 infected cells. Using a biochemical assay to monitor HIV-1 CA core disassembly in infected cells, an increase was observed in amounts of intact (pelletable) CA cores upon DHC depletion or p50 over-expression. Results from these two complementary assays suggest that inhibiting dynein-mediated transport interferes with HIV-1 uncoating in infected cells, indicating the existence of a functional link between HIV-1 transport and uncoating (Pawlica, 2014).

Search PubMed for articles about Drosophila Dynamitin

Chatin, B., Colombier, P., Gamblin, A. L., Allouis, M., Le Bouffant, F. (2014). Dynamitin affects cell-surface expression of voltage-gated sodium channel Nav1.5. Biochem J, 463(3):339-349 PubMed ID: 25088759

Cheong, F. K., Feng, L., Sarkeshik, A., Yates, J. R., Schroer, T. A. (2014). Dynactin integrity depends upon direct binding of dynamitin to Arp1. Mol Biol Cell, 25(14):2171-2180 PubMed ID: 24829381

Hammesfahr, B., Kollmar, M. (2012). Evolution of the eukaryotic dynactin complex, the activator of cytoplasmic dynein. BMC Evol Biol, 12:95 PubMed ID: 22726940

Liu, G., Sanghavi, P., Bollinger, K. E., Perry, L., Marshall, B., Roon, P., Tanaka, T., Nakamura, A. and Gonsalvez, G. B. (2015). Efficient endocytic uptake and maturation in Drosophila oocytes requires Dynamitin/p50. Genetics 201(2):631-49. PubMed ID: 26265702

Ozaki, Y., Matsui, H., Nagamachi, A., Asou, H., Aki, D., Inaba, T. (2011). The dynactin complex maintains the integrity of metaphasic centrosomes to ensure transition to anaphase. J Biol Chem, 286(7):5589-5598 PubMed ID: 21163948

Pawlica, P., Berthoux, L. (2014). Cytoplasmic dynein promotes HIV-1 uncoating. Viruses, 6(11):4195-4211 PubMed ID: 25375884

Shen, C., Honda, H., Suzuki, S. O., Maeda, N., Shijo, M., Hamasaki, H., Sasagasako, N., Fujii, N., Iwaki, T. (2018). Dynactin is involved in Lewy body pathology. Neuropathology, 38(6):583-590 PubMed ID: 30215870

Whited, J. L., Cassell, A., Brouillette, M. and Garrity, P. A. (2004). Dynactin is required to maintain nuclear position within postmitotic Drosophila photoreceptor neurons. Development 131(19): 4677-4686. PubMed ID: 15329347

Wilkie, G. S. and Davis, I. (2001). Drosophila wingless and pair-rule transcripts localize apically by dynein-mediated transport of RNA particles. Cell 105(2): 209-219. PubMed ID: 11336671

Wu, C. H., Zong, Q., Du, A. L., Zhang, W., Yao, H. C., Yu, X. Q. and Wang, Y. F. (2016). Knockdown of Dynamitin in testes significantly decreased male fertility in Drosophila melanogaster. Dev Biol 420: 79-89. PubMed ID: 27742209

Zheng, Y., Wang, J. L., Liu, C., Wang, C. P., Walker, T. and Wang, Y. F. (2011). Differentially expressed profiles in the larval testes of Wolbachia infected and uninfected Drosophila. BMC Genomics 12: 595. PubMed ID: 22145623

date revised: 10 April 2026

Home page: The Interactive Fly © 2026 Thomas Brody, Ph.D.