Dynamics of cytoplasmic dynein in living cells and the effect of a mutation in the dynactin complex actin-related protein Arp1

Xiang, Xin; Han, Gongshe; Winkelmann, Donald A.; Zuo, Wenqi; Morris, Nicholas P.

doi:10.1016/s0960-9822(00)00488-7

Cited by 101 publications

(103 citation statements)

References 21 publications

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“…The spherical structures were reminiscent of endomembrane organelles that have been described in previous studies (Bowman et al 2009). The short linear tracks resembled the comet tail tip accumulation of dynein at microtubule plus ends that has been reported by studies in other systems ranging from fungi to mammalian neurons (Xiang et al 1995;Vaughan et al 1999;Xiang et al 2000;Han et al 2001;Ma and Chisholm 2002;Lee et al 2003;Sheeman et al 2003;Zhang et al 2003Zhang et al , 2010Zhang et al , 2011Lenz et al 2006;Arimoto et al 2011;Schuster et al 2011a). Apart from the spherical structures and short linear tracks, no additional substructures were visible upon standard exposure settings or photobleaching the bright hyphal tip dynein fluorescence ( Figure S3).…”

Section: Resultssupporting

confidence: 66%

“…This accumulation most likely represents a reservoir of dynein being retained at the hyphal tip for future cargo transport. In addition, wild-type dynein was also found on the ends of microtubules near the hyphal apex in the classic comet-tail structures described in a variety of past studies (Xiang et al 1995(Xiang et al , 2000Vaughan et al 1999;Han et al 2001;Ma and Chisholm 2002;Zhang et al 2003Zhang et al , 2010Zhang et al , 2011Lenz et al 2006;Arimoto et al 2011;Schuster et al 2011a).…”

Section: Discussionmentioning

confidence: 79%

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Analyses of Dynein Heavy Chain Mutations Reveal Complex Interactions Between Dynein Motor Domains and Cellular Dynein Functions

et al. 2012

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Cytoplasmic dynein transports cargoes for a variety of crucial cellular functions. However, since dynein is essential in most eukaryotic organisms, the in-depth study of the cellular function of dynein via genetic analysis of dynein mutations has not been practical. Here, we identify and characterize 34 different dynein heavy chain mutations using a genetic screen of the ascomycete fungus Neurospora crassa, in which dynein is nonessential. Interestingly, our studies show that these mutations segregate into five different classes based on the in vivo localization of the mutated dynein motors. Furthermore, we have determined that the different classes of dynein mutations alter vesicle trafficking, microtubule organization, and nuclear distribution in distinct ways and require dynactin to different extents. In addition, biochemical analyses of dynein from one mutant strain show a strong correlation between its in vitro biochemical properties and the aberrant intracellular function of that altered dynein. When the mutations were mapped to the published dynein crystal structure, we found that the three-dimensional structural locations of the heavy chain mutations were linked to particular classes of altered dynein functions observed in cells. Together, our data indicate that the five classes of dynein mutations represent the entrapment of dynein at five separate points in the dynein mechanochemical and transport cycles. We have developed N. crassa as a model system where we can dissect the complexities of dynein structure, function, and interaction with other proteins with genetic, biochemical, and cell biological studies.T HE organization, survival, and function of eukaryotic cells depend on intracellular transport governed by the microtubule-based molecular motors cytoplasmic dynein and kinesin. Dynein carries out the inward transport of cargos whereas kinesins are responsible for the outward movement. These motors, in addition to the transport and distribution of a wide variety of cargos, are also responsible for vital cellular processes ranging from mitosis to organelle positioning to embryonic development (Schroer et al. 1989;Burkhardt et al. 1997;Rana et al. 2004;Kardon and Vale 2009). Although intracellular transport is necessary for the function of all cells, polarized cells in particular have specific transport needs due to their asymmetry and elongated shape. These extraordinary requirements necessitate efficient longrange microtubule-based transport mechanisms (Hirokawa and Takemura 2005;Zheng et al. 2008;Harada 2010). The anterograde transport needs in these cells are satisfied by a variety of kinesins but only a single cytoplasmic dynein fulfills the retrograde transport requirements.Cytoplasmic dynein is a megadalton-sized, multiprotein complex composed of two heavy chains (DHCs), and varying numbers of dynein intermediate chains (DICs), light intermediate chains, and light chains. The DHCs perform the ATPase motor and microtubule-binding functions and the other subunits couple dynein to dynact...

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Section: Resultssupporting

confidence: 66%

Section: Discussionmentioning

confidence: 79%

Analyses of Dynein Heavy Chain Mutations Reveal Complex Interactions Between Dynein Motor Domains and Cellular Dynein Functions

et al. 2012

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“…In wild-type background, GFP-dynein forms comet-like structures representing its localization at the microtubule plus end (Xiang et al 2000;Han et al 2001). In some hyphae, small speckles along microtubules can also be found, but it is hard to track them because of the low intensity and the transient nature of these signals.…”

Section: Discussionmentioning

confidence: 99%

“…Construction of a GFP-nudA strain in which the GFP-nudA fusion is under the control of its own promoter: We have previously constructed a strain in which GFP is inserted in the nudA coding region between the eighth and the ninth aa and the GFP-nudA fusion is under the control of the alcA promoter (Xiang et al 2000). In medium containing glucose, the expression of the GFP-nudA fusion is repressed, and our previous observations of GFP-NUDA were all done using medium containing glycerol as the carbon source.…”

Section: Strains and Aspergillus Techniquesmentioning

confidence: 99%

“…To make this new strain, a 1.5-kb fragment upstream of the nudA coding sequence was amplified from genomic DNA of the wildtype strain GR5 by using the following primers: NudAupper (59-AGTAAGCCAAGTTTCTACCGAACG-39) and NudAlower (59-TTCGCGGGCAGATAGAGTTTTCCCGCGACGCTGGTC CGTAAC-39). A 2.3-kb SmaI and BamHI fragment was obtained from the original alcA-GFP-nudA plasmid (Xiang et al 2000). Subsequently, a fusion PCR was performed to fuse the 1.5-and 2.3-kb fragments with the following primers: NudAupper (59-AGTAAGCCAAGTTTCTACCGAACG-39) and NudAend (59-CTTCATTAAGGTGCGGAATTCGCG-39).…”

Section: Strains and Aspergillus Techniquesmentioning

confidence: 99%

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Point Mutations in the Stem Region and the Fourth AAA Domain of Cytoplasmic Dynein Heavy Chain Partially Suppress the Phenotype of NUDF/LIS1 Loss in Aspergillus nidulans

Zhuang

Zhang²,

Xiang

2007

Genetics

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Cytoplasmic dynein performs multiple cellular tasks but its regulation remains unclear. The dynein heavy chain has a N-terminal stem that binds to other subunits and a C-terminal motor unit that contains six AAA (ATPase associated with cellular activities) domains and a microtubule-binding site located between AAA4 and AAA5. In Aspergillus nidulans, NUDF (a LIS1 homolog) functions in the dynein pathway, and two nudF6 partial suppressors were mapped to the nudA dynein heavy chain locus. Here we identified these two mutations. The nudAL1098F mutation resides in the stem region, and nudAR3086C is in the end of AAA4. These mutations partially suppress the phenotype of nudF deletion but do not suppress the phenotype exhibited by mutants of dynein intermediate chain and Arp1. Surprisingly, the stronger DnudF suppressor, nudAR3086C, causes an obvious decrease in the basal level of dynein's ATPase activity and an increase in dynein's distribution along microtubules. Thus, suppression of the DnudF phenotype may result from mechanisms other than simply the enhancement of dynein's ATPase activity. The fact that a mutation in the end of AAA4 negatively regulates dynein's ATPase activity but partially compensates for NUDF loss indicates the importance of the AAA4 domain in dynein regulation in vivo.

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The yeast ubiquitin‐like domain protein Mdy2 is required for microtubule‐directed nuclear migration and localizes to cytoplasmic granules in response to heat stress

et al. 2010

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MDY2 encodes a ubiquitin-like (UBL)-domain protein necessary for efficient mating in Saccharomyces cerevisiae. Unlike most UBL proteins, Mdy2 is apparently not subject to C-terminal processing and is localized predominantly in the nucleus. Deletion of MDY2 is associated with a five- to seven-fold reduction in mating efficiency, mainly due to defects in nuclear migration and karyogamy at the prezygotic stage. Here, we looked for two potential interacting partners of Mdy2, investigated the function of Mdy2 in nuclear movement, determined the increased heat sensitivity defects of mdy2Δ mutants, and inspected localization of Mdy2. Coprecipitation studies show that Mdy2 associates with α-tubulin and with the microtubule (MT)-associated dynactin subunit p150(Glued)/Nip100. nip100Δ mutants exhibit no defects in nuclear migration or in MT length or orientation during shmooing growth. Deletion of MDY2 display small nuclear migration phenotype during vegetative growth and seems to exacerbate the defects in mitotic nuclear migration seen in the nip100Δ strain. Deletion of MDY2 increased heat sensitivity of the cells and these strains accumulate mitotic nuclear migration defects and shortened MTs under these conditions. GFP-Mdy2 proteins which are localized predominantly in the nucleus at permissive temperature are localized to cytoplasmic foci during heat shock. Colocalization studies revealed that heat stress-induced enrichment of Mdy2 in cytoplasmic foci merged mainly with stress granules marker Pab1. During glucose deprivation a minority of Mdy2 foci overlapped with P-bodies marker Dcp2, while most Mdy2 foci and Pab1 foci overlap. Accordingly, we propose that Mdy2 plays a critical role in the MT-dependent processes of karyogamy and stress response.

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Dynamics of cytoplasmic dynein in living cells and the effect of a mutation in the dynactin complex actin-related protein Arp1

Cited by 101 publications

References 21 publications

Analyses of Dynein Heavy Chain Mutations Reveal Complex Interactions Between Dynein Motor Domains and Cellular Dynein Functions

Analyses of Dynein Heavy Chain Mutations Reveal Complex Interactions Between Dynein Motor Domains and Cellular Dynein Functions

Point Mutations in the Stem Region and the Fourth AAA Domain of Cytoplasmic Dynein Heavy Chain Partially Suppress the Phenotype of NUDF/LIS1 Loss in Aspergillus nidulans

The yeast ubiquitin‐like domain protein Mdy2 is required for microtubule‐directed nuclear migration and localizes to cytoplasmic granules in response to heat stress

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