Actin-binding proteins from Drosophila embryos: a complex network of interacting proteins detected by F-actin affinity chromatography.

Miller, Ken; Field, Christine M.; Alberts, Bruce

doi:10.1083/jcb.109.6.2963

Cited by 155 publications

(131 citation statements)

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“…Electrophoresis was carried out with a 4-20% polyacrylamide gradient gel (BioRad) in SDS running buffer. Proteins were wet-transferred to a 0.45-μm nylon membrane, and the membrane was probed with the respective antibodies in 5% milk TBST using the following dilutions: P4D2 anti-Piwi (1:66) (10), 4D10 anti-Aub (1:1,000) (35), and 3C7 anti-myosin VI (1:20) (47). HRP (horseradish peroxidase) conjugated secondary antibodies (KPL) and substrates (Millipore) were used according to vendors' instructions to visualize the results.…”

Section: Methodsmentioning

confidence: 99%

Drosophila Piwi functions downstream of piRNA production mediating a chromatin-based transposon silencing mechanism in female germ line

Wang

Elgin

2011

Proc. Natl. Acad. Sci. U.S.A.

209

232

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Transposon control is a critical process during reproduction. The PIWI family proteins can play a key role, using a piRNA-mediated slicing mechanism to suppress transposon activity posttranscriptionally. In Drosophila melanogaster , Piwi is predominantly localized in the nucleus and has been implicated in heterochromatin formation. Here, we use female germ-line–specific depletion to study Piwi function. This depletion of Piwi leads to infertility and to axis specification defects in the developing egg chambers; correspondingly, widespread loss of transposon silencing is observed. Germ-line Piwi does not appear to be required for piRNA production. Instead, Piwi requires Aubergine (and presumably secondary piRNA) for proper localization. A subset of transposons that show significant overexpression in germ-line Piwi-depleted ovaries exhibit a corresponding loss of HP1a and H3K9me2. Germ-line HP1a depletion also leads to a loss of transposon silencing, demonstrating the functional requirement for HP1a enrichment at these loci. Considering our results and those of others, we infer that germ-line Piwi functions downstream of piRNA production to promote silencing of some transposons via recruitment of HP1a. Thus, in addition to its better-known function in posttranscriptional silencing, piRNA also appears to function in a targeting mechanism for heterochromatin formation mediated by Piwi.

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Section: Methodsmentioning

confidence: 99%

Drosophila Piwi functions downstream of piRNA production mediating a chromatin-based transposon silencing mechanism in female germ line

Wang

Elgin

2011

Proc. Natl. Acad. Sci. U.S.A.

209

232

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“…Although a large number of actin-binding proteins have been found, there remain many, particularly those of low abundance, that remain undiscovered [1,[9][10][11][12][13][14]. Furthermore, it is still not understood how those proteins that have been identified interact to produce even one of the many aspects of platelet behavior.…”

Section: Introductionmentioning

confidence: 99%

“…Those actin-binding proteins that have already been characterized can be eliminated from study either by their molecular weight or by Western blotting with antibodies to them, thus uncovering any novel, less abundant proteins likely to be isolated by this affinity approach. This technique has successfully identified large numbers of actin-binding proteins in other systems [11][12][13][14]. In yeast and Drosophila, where only a few actin-binding proteins were previously identified, some of these proteins were subsequently found to be homologues of known actin-binding proteins, while many others appear to be novel [17][18][19].…”

Section: Introductionmentioning

confidence: 99%

“…In yeast and Drosophila, where only a few actin-binding proteins were previously identified, some of these proteins were subsequently found to be homologues of known actin-binding proteins, while many others appear to be novel [17][18][19]. Immunofluorescence permitted preliminary assignment of each of these proteins to particular subcellular structures, which aids in focusing subsequent studies on their functional role [11][12][13]. A platelet protein identified by F-actin affinity columns, the Mab 2E4 antigen, colocalizes with actin in the lamellipodium of migrating cells and nerve growth cones, and binds the ends of actin filaments in vitro [20,21], but is not significantly homologous to any other actin-binding protein (Bearer, manuscript in preparation).…”

Section: Introductionmentioning

confidence: 99%

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Cytoskeletal domains in the activated platelet

Bearer

1995

Cell Motil. Cytoskeleton

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Platelets circulate in the blood as discoid cells which, when activated, change shape by polymerizing actin into various structures, such as filopodia and stress fibers. In order to understand this process, it is necessary to determine how many other proteins are involved. As a first step in defining the full complement of actin-binding proteins in platelets, filamentous (F)-actin affinity chromatography was used. This approach identified >30 different proteins from ADP-activated human blood platelets which represented 4% of soluble protein. Although a number of these proteins are previously identified platelet actin-binding proteins, many others appeared to be novel. Fourteen different polyclonal antibodies were raised against these apparently novel proteins and used to sort them into nine categories based on their molecular weights and on their location in the sarcomere of striated muscle, in fibroblasts and in spreading platelets. Ninetythree percent of these proteins (13 of 14 proteins tested) were found to be associated with actinrich structures in vivo.Four distinct actin filament structures were found to form during the initial 15 min of activation on glass: filopodia, lamellipodia, a contractile ring encircling degranulating granules, and thick bundles of filaments resembling stress fibers. Actin-binding proteins not localized in the discoid cell became highly concentrated in one or another of these actin-based structures during spreading, such that each structure contains a different complement of proteins. These results present crucial information about the complexity of the platelet cytoskeleton, demonstrating that four different actin-based structures form during the first 15 min of surface activation, and that there remain many as yet uncharacterized proteins awaiting further investigation that are differentially involved in this process.

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“…In addition, several other known cortical components of the classical cytokinesis contractile rings are found concentrated within the metaphase furrows and the furrow canals of cellularizing embryos (Zalokar and Erk, 1976;Foe and Alberts, 1983). These include myosin II (Kiehart, 1990;Young et al, 1991;Field and Alberts, 1995;Foe et al, 2000), anillin (Miller et al, 1989;Field and Alberts, 1995), the formins (Afshar et al, 2000), and the septins (Neufeld and Rubin, 1994;Fares et al, 1995;Field et al, 1996). However, the specific roles played by these other cortical components during these membrane invaginations are still unclear.…”

mentioning

confidence: 99%

Reassessing the Role and Dynamics of Nonmuscle Myosin II during Furrow Formation in EarlyDrosophilaEmbryos

Royou

Field

Sisson

et al. 2004

MBoC

280

293

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The early Drosophila embryo undergoes two distinct membrane invagination events believed to be mechanistically related to cytokinesis: metaphase furrow formation and cellularization. Both involve actin cytoskeleton rearrangements, and both have myosin II at or near the forming furrow. Actin and myosin are thought to provide the force driving membrane invagination; however, membrane addition is also important. We have examined the role of myosin during these events in living embryos, with a fully functional myosin regulatory light-chain-GFP chimera. We find that furrow invagination during metaphase and cellularization occurs even when myosin activity has been experimentally perturbed. In contrast, the basal closure of the cellularization furrows and the first cytokinesis after cellularization are highly dependent on myosin. Strikingly, when ingression of the cellularization furrow is experimentally inhibited by colchicine treatment, basal closure still occurs at the appropriate time, suggesting that it is regulated independently of earlier cellularization events. We have also identified a previously unrecognized reservoir of particulate myosin that is recruited basally into the invaginating furrow in a microfilament-independent and microtubule-dependent manner. We suggest that cellularization can be divided into two distinct processes: furrow ingression, driven by microtubule mediated vesicle delivery, and basal closure, which is mediated by actin/myosin based constriction. INTRODUCTIONCytokinesis is the final step of cell division, required for the equal partitioning of the newly separated genetic material into two sister cells. Conventional animal cell cytokinesis is characterized by the assembly of an actin-myosin-based structure called the contractile ring, positioned midway between the two spindle poles, which drives the formation of a cleavage furrow. Despite its importance, many aspects of cytokinesis remain obscure. For example, although it is now well established that the cleavage plane can be specified by the central spindle or by astral microtubules (reviewed in Glotzer, 2001;Straight and Field, 2000), it is not known how microtubules trigger the assembly of the contractile ring or if they are required for regulating the contractile force.During the early phase of Drosophila embryogenesis, the embryo undergoes 13 rounds of nuclear division in the absence of cytokinesis. However, two kinds of membrane invagination events believed to be mechanistically related to conventional cytokinesis take place: metaphase furrow formation and cellularization. Metaphase furrows, or pseudocleavage furrows, form transiently during the 10th to 13th mitotic cycles of the cortical syncytial divisions. At interphase, actin is concentrated in cortical caps above each nucleus, but with entry into mitosis, the actin cytoskeleton reorganizes from caps to rings at the base of shallow furrows surrounding each developing spindle (Zalokar and Erk, 1976;Foe and Alberts, 1983;Karr and Alberts, 1986;Foe et al., 2000). These furrows fo...

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Actin-binding proteins from Drosophila embryos: a complex network of interacting proteins detected by F-actin affinity chromatography.

Cited by 155 publications

References 19 publications

Drosophila Piwi functions downstream of piRNA production mediating a chromatin-based transposon silencing mechanism in female germ line

Drosophila Piwi functions downstream of piRNA production mediating a chromatin-based transposon silencing mechanism in female germ line

Cytoskeletal domains in the activated platelet

Reassessing the Role and Dynamics of Nonmuscle Myosin II during Furrow Formation in EarlyDrosophilaEmbryos

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