An actomyosin clamp assembled by the Amphiphysin-Rho1-Dia/DAAM-Rok pathway reinforces somatic cell membrane folded around spermatid heads

Kapoor, Tushna; Dubey, P K; Shirolikar, Seema; Ray, Krishanu

doi:10.1016/j.celrep.2021.108918

Cited by 4 publications

(16 citation statements)

References 46 publications

(71 reference statements)

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“…The process of spermatid maturation and release in Drosophila testis provides an excellent opportunity to study the molecular cell biology of the interplay between F-actin dynamics and membrane sculpting at the tissue and organ level ex vivo . For example, the indentation of the head cyst cell membrane by mature spermatids was shown to induce two different forms of F-actin assembly at distinct time scales using time-lapse confocal imaging from isolated Drosophila testis ( Dubey et al., 2016 )( Kapoor et al., 2021 ). This system provided an opportunity to observe subcellular changes in the F-actin dynamics and correlate them with the morphogenetic processes in different genetic backgrounds and in the presence of specific function-blocking reagents, using an inexpensive and straightforward protocol.…”

Section: Before You Beginmentioning

confidence: 99%

“…The Protamine complex replaces Histones during spermatid maturation and labels the mature spermatid heads. Using this protocol, we have also imaged the morphogenesis of septate junctions (SJs) using neuregulin-GFP protein trap transgenic line ( Nrg-GFP ), which marks the SJs between the cyst cells ( Dubey et al., 2019 ), and the myosin motor localization and dynamics at the actin cap using the GFP-tagged myosin light chain ( squash-GFP ) transgene expressed under its own promoter ( sqh::sqh-GFP ) ( Kapoor et al., 2021 ). The same protocol is used to describe sperm release ( Dubey et al., 2016 ) and the spermatid tail coiling (this study) using DonJuan-GFP ( DJ-GFP ), which marks the mitochondrial derivatives along the sperm tail.…”

Section: Before You Beginmentioning

confidence: 99%

“… (E) The TE zone of a testis from the ECadh-GFP protein trap adult expressing E-cadherin-GFP fusion protein under the endogenous promoter stained with the Hoechst dye (red) and FITC-Phalloidin (cyan). The arrow indicates NB, and the head cyst cell (HCC) boundary is indicated by a white dashed line (for detail, see Kapoor et al., 2021 ) …”

Section: Before You Beginmentioning

confidence: 99%

“…During early individualization stages, the NB is conically arranged (arrowhead, Figure 2 A), and the early individualization complex is present just caudal to the NB (arrow, Figure 2 A; Methods video S5 A) ( Desai et al, 2009 ). As the individualization complex proceeds, the NB becomes parallelly arranged, and over time, actin accumulation around the NB could be seen ( Desai et al, 2009 ; Kapoor et al., 2021 ). During this period, the spermatid tails (identified by DJ-GFP ) coil (arrow, Figure 2 B; Methods video S5 B) and the entire cyst assembly moves into the terminal epithelium (TE) zone.…”

Section: Expected Outcomesmentioning

confidence: 99%

“…

Figure 2 Capturing gross morphological changes during the individualization-to-coiling transition (A) Long-term confocal imaging of sqh::utr-GFP/+; protB-dsRed/+ testes revealed that the progression of F-actin cones (green, arrow) coincides with the NB compaction (red, arrowhead). Please see Methods video S5 A and Kapoor et al. (2021) for more detail.…”

Section: Expected Outcomesmentioning

confidence: 99%

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Time-lapse imaging of Drosophila testis for monitoring actin dynamics and sperm release

2022

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Summary Here we describe a simple step-by-step protocol for collecting high-resolution, time-lapse images of intact Drosophila testis ex vivo for a limited period using a confocal microscope, with minimum photo-toxic damage, to monitor spermatid individualization, coiling, and release. The F-actin dynamics during spermatid morphogenesis can be further investigated through laser ablations, Fluorescence-Recovery-After-Photobleaching, and drug treatments, using this protocol. For complete details on the use and execution of this protocol, please refer to Dubey et al. (2016) , Dubey et al. (2019) , and Kapoor et al. (2021) .

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Section: Before You Beginmentioning

confidence: 99%

Section: Before You Beginmentioning

confidence: 99%

Section: Before You Beginmentioning

confidence: 99%

Section: Expected Outcomesmentioning

confidence: 99%

“…

Section: Expected Outcomesmentioning

confidence: 99%

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Time-lapse imaging of Drosophila testis for monitoring actin dynamics and sperm release

2022

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Expression Analysis in a Dispersal-Fecundity Polyphenism Identifies Growth Regulators and Effectors

Angelini

Steele

Yorsz

et al. 2022

Integrative and Comparative Biology

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Polyphenism allows organisms to respond to varying environmental conditions by adopting alternative collections of morphological traits, often leading to different reproductive strategies. In many insects, polyphenism affecting the development of flight trades dispersal ability for increased fecundity. The soapberry bug Jadera haematoloma (Hemiptera: Rhopalidae) exhibits wing polyphenism in response to juvenile nutritional resources and cohort density. Development of full-length wings and flight-capable thoracic muscles occurs more frequently in cohorts raised under low food density conditions, and these features are correlated to reduced female fecundity. Soapberry bugs represent an example of polyphenic dispersal-fecundity trade-off. Short-wing development is not sex-limited, and morphs can also differ in male fertility. We have previously shown, via a candidate gene approach, that manipulation of insulin signaling can alter the threshold for nutritional response and that changes in the activity of this pathway underlie, at least in part, differences in the polyphenic thresholds in different host-adapted populations of J. haematoloma. We now expand the examination of this system using transcriptome sequencing across a multidimensional matrix of life stage, tissue, sex, food density and host population. We also examine the use of wing and thorax shape as factors modeling gene expression. In addition to insulin signaling, we find that components of the TOR, Hippo, Toll and estrogen-related receptor pathways are differentially expressed in the thorax of polyphenic morphs. The transcription factor Sox14 was one of the few genes differentially expressed in the gonads of morphs, being up-regulated in ovaries. We identify two transcription factors as potential mediators of morph-specific male fertility differences. We also find that bugs respond to nutrient limitation with expression of genes linked to cuticle structure and spermatogenesis. These findings provide a broad perspective from which to view this nutrition-dependent polyphenism.

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Emergent dynamics of adult stem cell lineages from single nucleus and single cell RNA-Seq ofDrosophilatestes

Raz

Vida²,

Stern

et al. 2022

Preprint

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Proper differentiation of sperm from germline stem cells, essential for production of the next generation, requires dramatic changes in gene expression that drive remodeling of almost all cellular components, from chromatin to organelles to cell shape itself. Here we provide a single nucleus and single cell RNA-seq resource covering all of spermatogenesis in Drosophila starting from in-depth analysis of adult testis single nucleus RNA-seq (snRNA-seq) data from the Fly Cell Atlas (FCA) study (Li et al., 2022). With over 44,000 nuclei and 6,000 cells analyzed, the data provide identification of rare cell types, mapping of intermediate steps in differentiation, and the potential to identify new factors impacting fertility or controlling differentiation of germline and supporting somatic cells. We justify assignment of key germline and somatic cell types using combinations of known markers, in situ hybridization, and analysis of extant protein traps. Comparison of single cell and single nucleus datasets proved particularly revealing of dynamic developmental transitions in germline differentiation. To complement the web-based portals for data analysis hosted by the FCA, we provide datasets compatible with commonly used software such as Seurat and Monocle. The foundation provided here will enable communities studying spermatogenesis to interrogate the datasets to identify candidate genes to test for function in vivo.

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An actomyosin clamp assembled by the Amphiphysin-Rho1-Dia/DAAM-Rok pathway reinforces somatic cell membrane folded around spermatid heads

Cited by 4 publications

References 46 publications

Time-lapse imaging of Drosophila testis for monitoring actin dynamics and sperm release

Time-lapse imaging of Drosophila testis for monitoring actin dynamics and sperm release

Expression Analysis in a Dispersal-Fecundity Polyphenism Identifies Growth Regulators and Effectors

Emergent dynamics of adult stem cell lineages from single nucleus and single cell RNA-Seq ofDrosophilatestes

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