Prominent Actin Fiber Arrays inDrosophilaTendon Cells Represent Architectural Elements Different from Stress Fibers

Functional and Genetic Analysis of Spectraplakins in Drosophila

“…In particular, the newly introduced use of small shRNAs, as compared to long double-stranded RNA, makes it now possible to generate rescue constructs with codon modifications within the targeted sequence that are immune to the knockdown. There are various sources of shot RNAi lines: Subramanian et al successfully used the UAS-dskakRNA line which reproduced shot mutant phenotypes in tendon cells and the nervous system (Alves-Silva et al, 2008Subramanian et al, 2003). A second-generation shot TRiP line (GL01286; Bloomington stock #41858) was shown to reduce shot transcript levels in the nervous system when expressed via elav-GAL4 (Valakh et al, 2013).…”

“…Drosophila has an exoskeleton in the form of an extracellular matrix (ECM) called cuticle which is secreted from the apical surface of the epidermis (Broadie, Baumgartner, & Prokop, 2011), whereas muscle tips anchor to the opposite basal surface of the epidermis via integrin-and ECM-dependent junctions (Prokop, Martín-Bermudo, Bate, & Brown, 1998). To withstand the enormous muscular pulling forces, the epidermis forms specialized tendon cells at these anchor points which contain dense arrays of actin and MT filaments linking the basally attached muscles to the apical cuticle on the other side (Alves-Silva et al, 2008). This organization is functionally analogous to basal keratinocytes where keratin filament networks link basal integrindependent hemidesmosomes to desmosomes on lateral and apical surfaces of the same cells (Aumailley et al, 2006).…”

“…Visualization is further facilitated by markers that can be used to specifically label tendon cells. These markers include endogenous proteins, such as integrins and their associated adhesion complex components; ECM proteins such as Tiggrin and Thrombospondin; the transcription factors Stripe, F-actin which is highly enriched in tendon cells, or the β1-tubulin isoform which is exclusive to the nervous system and tendon cells (Alves-Silva et al, 2008;Buttgereit, Leiss, Michiels, & Renkawitz-Pohl, 1991;Fogerty et al, 1994;Subramanian, Wayburn, Bunch, & Volk, 2007;Volohonsky, Edenfeld, Klambt, & Volk, 2007). For the detection of these proteins, antibodies or genomically GFP-tagged gene versions (Section 2.3) can be used, and also phalloidin works extremely well (Alves-Silva et al, 2008).…”

“…These markers include endogenous proteins, such as integrins and their associated adhesion complex components; ECM proteins such as Tiggrin and Thrombospondin; the transcription factors Stripe, F-actin which is highly enriched in tendon cells, or the β1-tubulin isoform which is exclusive to the nervous system and tendon cells (Alves-Silva et al, 2008;Buttgereit, Leiss, Michiels, & Renkawitz-Pohl, 1991;Fogerty et al, 1994;Subramanian, Wayburn, Bunch, & Volk, 2007;Volohonsky, Edenfeld, Klambt, & Volk, 2007). For the detection of these proteins, antibodies or genomically GFP-tagged gene versions (Section 2.3) can be used, and also phalloidin works extremely well (Alves-Silva et al, 2008). In addition, stripe-GAL4 can be used to drive any gene constructs of choice specifically in tendon cells, for example, to label them or to perform structure-function analyses with shot deletion constructs in shot mutant embryos (Alves-Silva et al, 2008;Bottenberg et al, 2009).…”

“…For the detection of these proteins, antibodies or genomically GFP-tagged gene versions (Section 2.3) can be used, and also phalloidin works extremely well (Alves-Silva et al, 2008). In addition, stripe-GAL4 can be used to drive any gene constructs of choice specifically in tendon cells, for example, to label them or to perform structure-function analyses with shot deletion constructs in shot mutant embryos (Alves-Silva et al, 2008;Bottenberg et al, 2009). Specimens can be either analyzed live, cut open, and dissected flat or injected with a fixation/labeling solution containing paraformaldehyde, phalloidin, and Triton-X (Alves- Silva et al, 2008;Budnik et al, 2006;Prokop & Technau, 1993).…”