About

Amy Kiger received her Ph.D. as a Howard Hughes Medical Institute Predoctoral Fellow from the Department of Developmental Biology at Stanford School of Medicine. She then completed postdoctoral studies in the Department of Genetics at Harvard Medical School as a Fellow of the Jane Coffin Childs Memorial Fund for Medical Research. As a Principal Investigator, she leads her own research laboratory focused on cellular remodeling, membrane regulation, autophagy, and systems biology approaches to understanding cell shape and organization. Her research investigates how membrane trafficking processes, under the control of spatiotemporal lipid regulation, coordinate dynamic cell structures, with a foundation in genetic and cell biology experiments in Drosophila. Her work links specific lipid kinase and phosphatase activities to cell remodeling, aiming to identify conserved mechanisms of membrane regulation in immune and muscle cell functions, and to provide insights into related human diseases. Key areas of her research include phosphoinositide lipid regulation in cellular remodeling, the role of autophagy in cell shape changes, and the genetic networks controlling cellular morphology. Her contributions have advanced understanding of membrane trafficking, autophagy's role in cell shape, and the genetic regulation of cellular remodeling processes.

Research topics

• Cell biology
• Biochemistry
• Biology

Selected publications

• PI(4,5)P ₂ role in Transverse-tubule membrane formation and muscle function

  bioRxiv (Cold Spring Harbor Laboratory) · 2024-01-31 · 2 citations

  preprintOpen accessSenior authorCorresponding

  Abstract Transverse (T)-tubules – vast, tubulated domains of the muscle plasma membrane – are critical to maintain healthy skeletal and heart contractions. How the intricate T-tubule membranes are formed is not well understood, with challenges to systematically interrogate in muscle. We established the use of intact Drosophila larval body wall muscles as an ideal system to discover mechanisms that sculpt and maintain the T-tubule membrane network. A muscle-targeted genetic screen identified specific phosphoinositide lipid regulators necessary for T-tubule organization and muscle function. We show that a PI4KIIIα - Skittles/PIP5K pathway is needed for T-tubule localized PI(4)P to PI(4,5)P 2 synthesis, T-tubule organization, calcium regulation, and muscle and heart rate functions. Muscles deficient for PI4KIIIα or Amphiphysin , the homolog of human BIN1 , similarly exhibited specific loss of transversal T-tubule membranes and dyad junctions, yet retained longitudinal membranes and the associated dyads. Our results highlight the power of live muscle studies, uncovering distinct mechanisms and functions for sub-compartments of the T-tubule network relevant to human myopathy. Summary T-tubules – vast, tubulated domains of the muscle plasma membrane – are critical to maintain skeletal and heart contractions. Fujita et al . establish genetic screens and assays in intact Drosophila muscles that uncover PI(4,5)P 2 regulation critical for T-tubule maintenance and function. Key Findings PI4KIIIα is required for muscle T-tubule formation and larval mobility. A PI4KIIIα-Sktl pathway promotes PI(4)P and PI(4,5)P 2 function at T-tubules. PI4KIIIα is necessary for calcium dynamics and transversal but not longitudinal dyads. Disruption of PI(4,5)P 2 function in fly heart leads to fragmented T-tubules and abnormal heart rate.

  PublisherOA PDFDOI

• An autophagy-dependent tubular lysosomal network synchronizes degradative activity required for muscle remodeling

  Journal of Cell Science · 2020 · 26 citations

  • Biology
  • Cell biology
  • Biochemistry

  -deficient mutants, the efficiency of lysosomal tubulation correlated with the phenotypic severity in muscle remodeling. The lumen of the tubular network was continuous and homogeneous across a broad region of the remodeling muscle. Altogether, we revealed that the dynamic expansion of a tubular autolysosomal network synchronizes the abundant degradative activity required for developmentally regulated muscle remodeling.

  PublisherOA PDFDOI

• Filling the Cannabis Misuse Knowledge Gap for College Campus Health Teams

  American journal of health studies · 2020-10-23

  articleOpen access

  Missouri legalized medicinal marijuana in 2018, however, knowledge gaps related to cannabis misuse exist on college campuses. This study evaluated the effectiveness of professional development on cannabis misuse. A mixed-method design was used. Subjects included students, faculty, and staff (N=40). Using an independent samples t-test (p<.05), analysis of student results (n=33) demonstrated significant increases in confidence in identifying various cannabis forms, routes, and safe levels of use; explaining problems related to cannabis use; discussing cannabis use and decreasing cannabis use; and implications of law and workplace policies. Participants reported being likely to use or share what they learned. There was no effect on the intention to change their use of cannabis. A further need for cannabis-related pregnancy and pain treatment information was reported.

  PublisherDOI

• An Autophagy-Dependent Tubular Lysosomal Network Synchronizes Degradative Activity Required for Muscle Remodeling

  bioRxiv (Cold Spring Harbor Laboratory) · 2020 · 1 citations

  • Cell biology
  • Biology
  • Biochemistry

  Abstract Previously, we reported that autophagy is critical for Drosophila muscle remodeling during metamorphosis (Fujita et al., 2017). However, little is known about how lysosomes meet increased degradative demand upon cellular remodeling. Here, we found an extensive tubular autolysosomal network in remodeling muscle. The tubular network transiently appeared and exhibited the capacity to degrade autophagic cargoes. The tubular autolysosomal network was uniquely marked by the autophagic SNARE protein, Syntaxin 17, and its formation depended on both autophagic flux and degradative function, with the exception of the Atg12 and Atg8 ubiquitin-like conjugation systems. Among ATG -deficient mutants, the efficiency of lysosomal tubulation correlated with the phenotypic severity in muscle remodeling. The lumen of the tubular network was continuous and homogeneous across a broad region of the remodeling muscle. Altogether, we revealed that the dynamic expansion of a tubular autolysosomal network synchronizes the abundant degradative activity required for developmentally regulated muscle remodeling. Impact Statement Analysis of developmentally-regulated Drosophila muscle remodeling revealed autophagy-dependent formation of an extensive, Syntaxin 17-marked, tubular network that synchronizes the abundant degradative activity across a broad region of the remodeling muscle

  PublisherOA PDFDOI

• Genetic screen in Drosophila muscle identifies autophagy-mediated T-tubule remodeling and a Rab2 role in autophagy

  eLife · 2017-01-07 · 117 citations

  articleOpen accessSenior author

  Transverse (T)-tubules make-up a specialized network of tubulated muscle cell membranes involved in excitation-contraction coupling for power of contraction. Little is known about how T-tubules maintain highly organized structures and contacts throughout the contractile system despite the ongoing muscle remodeling that occurs with muscle atrophy, damage and aging. We uncovered an essential role for autophagy in T-tubule remodeling with genetic screens of a developmentally regulated remodeling program in Drosophila abdominal muscles. Here, we show that autophagy is both upregulated with and required for progression through T-tubule disassembly stages. Along with known mediators of autophagosome-lysosome fusion, our screens uncovered an unexpected shared role for Rab2 with a broadly conserved function in autophagic clearance. Rab2 localizes to autophagosomes and binds to HOPS complex members, suggesting a direct role in autophagosome tethering/fusion. Together, the high membrane flux with muscle remodeling permits unprecedented analysis both of T-tubule dynamics and fundamental trafficking mechanisms.

  PublisherDOI

• Author response: Genetic screen in Drosophila muscle identifies autophagy-mediated T-tubule remodeling and a Rab2 role in autophagy

  2016-12-17 · 2 citations

  peer-reviewOpen accessSenior author

  Article Figures and data Abstract Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Transverse (T)-tubules make-up a specialized network of tubulated muscle cell membranes involved in excitation-contraction coupling for power of contraction. Little is known about how T-tubules maintain highly organized structures and contacts throughout the contractile system despite the ongoing muscle remodeling that occurs with muscle atrophy, damage and aging. We uncovered an essential role for autophagy in T-tubule remodeling with genetic screens of a developmentally regulated remodeling program in Drosophila abdominal muscles. Here, we show that autophagy is both upregulated with and required for progression through T-tubule disassembly stages. Along with known mediators of autophagosome-lysosome fusion, our screens uncovered an unexpected shared role for Rab2 with a broadly conserved function in autophagic clearance. Rab2 localizes to autophagosomes and binds to HOPS complex members, suggesting a direct role in autophagosome tethering/fusion. Together, the high membrane flux with muscle remodeling permits unprecedented analysis both of T-tubule dynamics and fundamental trafficking mechanisms. https://doi.org/10.7554/eLife.23367.001 Introduction Differentiated muscle cells, or myofibers, are highly organized in order to coordinate the roles of specialized subcellular structures involved in contraction. Myofibril bundles of sarcomeres provide the contractile force. The power of contraction, however, requires synchronous sarcomere function under control of the 'excitation-contraction coupling' system that includes two membranous organelles, the sarcoplasmic reticulum (SR) and Transverse (T)-tubules (Al-Qusairi and Laporte, 2011). The T-tubule membrane network is continuous with the muscle cell plasma membrane, with tubulated membranes that invaginate radially inward in a repeated pattern at each sarcomere. With excitation-contraction coupling, neuromuscular action potentials are transmitted along the muscle T-tubule membrane to the SR junction, or dyad/triad, triggering coordinated SR Ca2+ release and synchronous sarcomere contractions (Al-Qusairi and Laporte, 2011). Formation of organized T-tubule membranes is thus critical for muscle function (Takeshima et al., 2015). Mechanisms must also remodel the T-tubule membrane network with ongoing myofiber reorganization in response to muscle use, damage, atrophy and aging. However, the extent and mechanisms of T-tubule remodeling remain largely unknown, in part due to challenges with observing T-tubule membrane network dynamics within intact mammalian myofibers. The T-tubule network includes both transversal and longitudinal tubular membrane elements that form and mature with myofiber differentiation and growth. In mouse skeletal muscle, mostly longitudinal tubular membranes initially present in embryonic muscle are remodeled postnatally with expansion to predominantly transversal tubular elements (Takekura et al., 2001). In contrast, both longitudinal and transversal T-tubule elements are maintained in adult mammalian cardiac muscle (Brette and Orchard, 2003) and in insect muscles (Razzaq et al., 2001). Relatively few molecular factors are known to shape the T-tubule network, and perhaps not surprisingly, all of which so far encode for membrane-associated functions (CAV3, DYSF, BIN1/Amph2, MTM1, DNM2) (Butler et al., 1997; Hnia et al., 2012; Lek et al., 2012; Morlot and Roux, 2013; Tang et al., 1996). Mutations in each also are associated with human myopathy and/or cardiomyopathy with T-tubule disorganization (Bashir et al., 1998; Betz et al., 2001; Bitoun et al., 2005; Laporte et al., 1996; Liu et al., 1998; Minetti et al., 1998; Nicot et al., 2007), pointing to the critical importance of membrane-mediated mechanisms to maintain the T-tubule membrane network. Drosophila is a powerful system for insights into the functional requirements for T-tubule formation and remodeling. The BIN1 BAR-domain protein has a conserved function involved in membrane tubulation required for T-tubule formation that was first described for the single Drosophila homolog, Amphiphysin (Lee et al., 2002b; Razzaq et al., 2001). The amph null mutant flies lack transversal T-tubule element membranes in myofibers at all developmental stages, corresponding with both larval and adult mobility defects (Razzaq et al., 2001). In contrast, the myotubularin (mtm) fly homolog of mammalian MTM1/MTMR2/MTMR1 subfamily of phosphatidylinositol 3-phosphate phosphatases is required only at later stages in development for T-tubule remodeling. While mtm loss of function has no obvious effects on larval muscle T-tubule organization or function, mtm-depleted post-larval stage muscles lack transversal T-tubule membranes with adult mobility defects in eclosion and flight (Ribeiro et al., 2011). Together, the amph and mtm mutant conditions that both lack transversal T-tubule elements in post-larval stage muscle yet different early development requirements underscores that distinct mechanisms are involved in T-tubule formation (amph-dependent) versus maintenance/remodeling (amph- and mtm-dependent). In Drosophila, a set of larval body wall muscles that persist as viable pupal abdominal muscles, called dorsal internal oblique muscles (IOMs), are essential for adult eclosion (Kimura and Truman, 1990). During metamorphosis, changes in IOM cell size and myofibril content have been noted (Kuleesha et al., 2014, 2016). We previously showed that wildtype IOMs undergo dramatic cortical and membrane remodeling with costamere integrin adhesion complex disassembly and reassembly at discrete pupal stages (Ribeiro et al., 2011). In contrast, the mtm-depleted IOMs exhibited persistent disassembly or a block in reassembly of integrin costameres along with the loss of transversal T-tubule membranes at late pupal stages, but without any precocious cell death (Ribeiro et al., 2011). A striking feature in the mtm-depleted IOMs was the accumulation of endosomal-like membranes decorated with integrin and T-tubule markers, Amph and Discs large (Dlg1, a PDZ protein). Altogether, these results suggest that T-tubule membranes may undergo disassembly-reassembly with normal myofiber remodeling, including the delivery of disassembled T-tubule membrane into an endomembrane trafficking pathway. The role for a molecular-cellular program in control of T-tubule remodeling that is at least partially distinct from that involved in initial T-tubule formation raises many questions about possible mechanisms, including the regulation of T-tubule organization and dynamics, the membrane fate(s) and source(s) with disassembly-reassembly, respectively, and the specific membrane trafficking routes and effectors involved. Possible hints may come from studies of other specialized dynamic cell membrane invaginations shown to involve endosomal and Golgi membrane trafficking pathways, such as cellularization of Drosophila syncytial embryos (Lee and Harris, 2013, 2014; Pelissier et al., 2003) and the tubulated demarcation membrane system in megakaryocyte platelet formation (Eckly et al., 2014). Membrane trafficking relies on the large family of Rab GTPases, with over sixty Rabs in humans and thirty in flies (Klöpper et al., 2012). The different Rabs are under distinct spatiotemporal regulation for recruitment, activation and functions at specific membrane compartments or domains. Guanine nucleotide exchange factors (GEFs) convert specific inactive GDP-bound Rabs to an active GTP-bound form. Active Rab-GTP then recruits a range of specific effector proteins to the membrane that mediate key trafficking functions, including cargo selection, vesicle budding, transport, tethering and fusion. Subsequently, GTPase-activating proteins (GAPs) deactivate Rabs by promoting GTP hydrolysis. Many membrane compartments have been defined by well-established localized functions of specific Rabs, for example: ER (Rab1), Golgi (Rab1, Rab6), secretory vesicles (Rab8), early endosomes (Rab5, Rab21), recycling endosomes (Rab11, Rab35), late endosomes (Rab7, Rab9), lysosomes (Rab7) and others (Jean and Kiger, 2012; Stenmark, 2009). Thus, identifying the specific Rabs required for a cellular process can provide clues to potential underlying membrane trafficking mechanisms involved. However, examples exist of Rabs with multiple known sites of function or yet unknown functions, and conversely, certain cellular processes – like T-tubule remodeling – lack defined roles yet for any Rabs. Here, we utilized the advantages of Drosophila IOMs to screen for Rab GTPases and related membrane trafficking functions required for T-tubule remodeling in intact muscle. Our results show that the entire contractile and excitation-contraction coupling system, including T-tubules, are disassembled and reassembled in IOMs during Drosophila metamorphosis. We found that autophagy, the membrane trafficking process for degradation of cytoplasmic contents by delivery to lysosomes, is upregulated with IOM remodeling where it plays an indispensable role for progression through T-tubule disassembly to reassembly. Our genetic analysis of IOM remodeling also reveals an unexpected and broad role for Rab2 in autophagy in flies and mammals. From our data, we propose that Rab2 localizes to autophagosomes where it interacts with the HOPS complex, which in turn, mediates tethering and trans-SNARE complex formation with Rab7-marked lysosomes to promote autophagosome-lysosome fusion. Together, these results show that Drosophila IOM remodeling provides an unprecedented in vivo context for discovery and analysis of T-tubule dynamics with relevance to human myopathy, as well as an ideal system due to high membrane flux to study fundamental trafficking pathways. Results Differentiated myofiber remodeling includes regulated T-tubule membrane disassembly and reassembly To monitor T-tubule remodeling in Drosophila abdominal muscles, we expressed the mCD8:GFP transmembrane fusion protein as a marker of the muscle cell and T-tubule membranes (Peterson and Krasnow, 2015). When observed in live IOM persistent larval muscles through the cuticle at 4 days after puparium formation (4d APF), mCD8:GFP showed a mesh-like pattern (Figure 1A) that in fixed samples colocalized with Dlg1-marked T-tubules (Figure 1B–D and Figure 1—figure supplement 1A) and sarcomere Z-lines (Figure 1—figure supplement 1B). The brightness of mCD8:GFP enabled us to monitor membrane dynamics during IOM remodeling in undissected live animals. As previously reported (Kuleesha et al., 2014; Wasser et al., 2007), the IOMs remodeled during metamorphosis with myofiber thinning through 2d APF followed by rethickening from 3-4d APF (Figure 1E–F, top and middle rows). Along with these cell morphology changes, the well-organized mCD8:GFP-marked membranes detected in third instar larval precursor muscles were disassembled in IOMs by 1-2d APF and then reassembled by 4d APF (Figure 1F, middle and bottom rows). Figure 1 with 1 supplement see all Download asset Open asset Detection of T-tubule membrane organization and remodeling in intact Internal Oblique Muscle (IOM) of live Drosophila. (A) mCD8:GFP showed a mesh-like pattern in pharate/pre-adult dorsal abdominal IOMs at 4d APF by live imaging, with both transversal and longitudinal membrane elements as indicated. (B) Schematic of IOM and z-section regions imaged in panel C. (C–D) Colocalization between mCD8:GFP (green) and Dlg1 (pink) at T-tubules in 4d APF IOMs quantified as Pearson's correlation between Dlg1 and GFP or mCD8:GFP; ± SEM of pooled data for 10 images from three experiments. (E) Time line of fly development from third instar larva to adult at 25°C; days after puparium formation (d APF). (F) Time course microscopy of mCD8:GFP in dorsal muscles imaged through the cuticle of live wildtype animals from third instar larva (3IL) to 4d APF, showing membrane remodeling in abdomens (top), central sections of individual IOMs (middle) and magnified view of boxed regions (bottom). See Figure 1—figure supplement 1 for related data. https://doi.org/10.7554/eLife.23367.002 Figure 1—source data 1 Relates to Figure 1D. Pearson correlation indicating colocalization between GFP or mCD8:GFP with Dlg1 at T-tubules in IOMs at 4d APF (.xlsx file). https://doi.org/10.7554/eLife.23367.003 Download elife-23367-fig1-data1-v2.xlsx To more specifically investigate myofiber remodeling, we monitored Dlg1:GFP (T-tubules), Reticulon:GFP (Sarcoplasmic Reticulum; SR), and GFP:actin (myofibrils) at 24h intervals during metamorphosis (Figure 2A). Each of these organelles was disassembled by 1-2d APF, and then reassembled by 4d APF in IOMs (Figure 2A). Furthermore, ultrastructual analysis of myofiber remodeling by transmission electron microscopy (TEM) imaging of IOM transverse sections (Figure 2B) confirmed both the timing and extent of the disassembly and reassembly stages during metamorphosis (Figure 2C–G). This reveals that differentiated myofiber structures critical for muscle function, including T-tubule membranes, undergo regulated and stereotypical remodeling in IOMs during metamorphosis. Figure 2 Download asset Open asset T-tubules disassemble and reassemble with IOM remodeling during metamorphosis. (A) Time course microscopy of Dlg1:GFP (T-tubule), Rtnl1:GFP (sarcoplasmic reticulum) or GFP:actin (myofibril) in wildtype animals at the indicated time points. (B) Schematic of an IOM TEM transverse section, as shown in 2C–G. (C–G) TEM images of IOM transverse sections in wildtype animals. Organized myofibrils and T-tubules were observed in both 3IL and 4d APF stages (C and G). At 1d APF, myofibrils were partially lost with mostly disorganized membranes (D). At 2d APF, myofibrils were completely absent with obvious appearance of autophagosomes and electron-dense lysosomal compartments (E). At 3d APF, myofibrils were reassembled but not well organized with a lack of obvious T-tubules (F). https://doi.org/10.7554/eLife.23367.005 A unique T-tubule remodeling phenotype with knockdown of a set of genes In order to identify functions involved in T-tubule membrane remodeling, we performed a muscle-targeted RNAi screen of candidate membrane trafficking-related genes, including all fly genes predicted to encode Rab GTPases, Arf GTPases, sorting nexins, BAR domain proteins, SNARE proteins, and phosphoinositide regulators (Supplementary file 1). Since T-tubule organization is required for muscle function (Al-Qusairi and Laporte, 2011; Razzaq et al., 2001), we first screened for muscle-targeted RNAi effects on fly mobility. Among 300 RNAi lines tested, 77 lines showed a defect in adult eclosion or mobility, 151 lines resulted in normal viability and mobility, and 83 lines were unscored due to pre-adult lethality (Figure 3A). As a secondary screen, we tested the 77 RNAi lines with eclosion or mobility defects for mCD8:GFP organization by live cell imaging in IOMs at 4d APF. We identified 10 RNAi lines targeting a set of 5 different genes, representing two Rab GTPases and three soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, with a similar phenotype (Figure 3A–B and Supplementary file 1). Instead of the organized T-tubule network seen in control IOMs, RNAi of Rab2, Rab7, Stx17, SNAP29 or Vamp7/8 each resulted in an accumulation of mCD8:GFP-positive small vesicles that filled often misshapen or swollen myofibers (Figure 3B). Unlike in controls, T-tubules (Dlg1) and most myofibrils (F-actin) were absent throughout these RNAi-treated IOMs at 4d APF (Figure 3C–D). Figure 3 with 1 supplement see all Download asset Open asset A unique T-tubule remodeling phenotype with knockdown of a set of known and unknown gene functions in autophagy. All IOMs imaged at 4d APF. (A) Muscle-targeted RNAi screen of IOM remodeling. In primary screen of 300 selected muscle-targeted RNAi lines (see text), 77 lines exhibited eclosion or adult mobility defects; these lines were used in a secondary screen for mCD8:GFP organization by confocal imaging. Three abnormal phenotype categories were identified for 37 lines. The shared 'small vesicle' phenotype was found for 10 RNAi lines for five genes presented here. (B) Rab2, Rab7, Stx17, SNAP29 or Vamp7/8 RNAi resulted in IOMs filled with small, mCD8:GFP-marked vesicles. Top row, brightly-marked dorsal IOMs in whole abdomen. Bottom row, magnified image of mCD8:GFP in single IOM. (C) Schematic of IOM and regions imaged in panel D–E. (D) T-tubule (Dlg1, green) and myofibril (F-actin, pink) organization in IOMs from control and Rab2, Rab7, Stx17, SNAP29, or Vamp7/8 RNAi conditions. (E) RNAi of HOPS components, Vps39, Vps18, or Vps11, exhibited shared phenotypes of (top) many mCD8:GFP-marked small vesicles and (bottom) lack of T-tubules (Dlg1, green) and myofibrils (F-actin, pink). See Figure 3—figure supplement 1 for related data. https://doi.org/10.7554/eLife.23367.006 Figure 3—source data 1 Relates to Figure 3—figure supplement 1B. Quantification of disorganized IOM mutant phenotypes observed from Rab2 RNAi co-expressed with lacZ negative control or with YFP:Rab2 wildtype rescue at 4d APF (.xlsx file). https://doi.org/10.7554/eLife.23367.007 Download elife-23367-fig3-data1-v2.xlsx The 'Rab2 class' of shared phenotypes suggested that these five genes all function in a shared process or pathway in IOMs. Indeed, it has been shown that four of the five genes play known roles together in lysosome fusion: Stx17, SNAP29, and Vamp7/8 form a trans-SNARE complex involved in autophagosome-lysosome fusion (Itakura et al., 2012; Takáts et al., 2013), while Rab7 functions in late endosome-lysosome fusion as well as late steps in autophagy (Gutierrez et al., 2004; Jäger et al., 2004; Hegedűs et al., 2016). It was unexpected, however, to find a shared RNAi phenotype between this set of known functions and Rab2, which had been implicated with functions at the ER and Golgi (Saraste, 2016). The specificity of the Rab2 RNAi phenotype was confirmed by rescue with co-expression of a wildtype Rab2 transgene (Figure 3—figure supplement 1A–B). From this, we speculated that the 'Rab2 class' of shared phenotypes was in each case a result of a block in autophagosome-lysosome fusion. To explore this possibility, we tested a requirement for the homotypic fusion and protein sorting (HOPS) complex that is known to mediate SNARE-dependent autophagosome-lysosome fusion (Jiang et al., 2014; Takáts et al., 2014). As expected, disruption of the HOPS complex with RNAi of subunits, Vps39, Vps18, and Vps11 each phenocopied the specific Rab2 class of IOM defects (Figure 3E and Figure 3—figure supplement 1C). These results suggest that a defect in autophagosome-lysosome fusion leads to the unique Rab2 class of RNAi phenotypes in IOM remodeling and predicts a novel role for Rab2 in autophagy. Rab2 or Rab7 knockdown blocks autophagosome-lysosome fusion with autophagy-dependent myofiber remodeling To address the underlying role for the 'Rab2 class' of genes in T-tubule remodeling, we characterized autophagy in IOMs with Rab2, Rab7 or Stx17 knockdown at 4d APF. The mCherry:GFP:Atg8a autophagic flux reporter indicates both dually-labeled Atg8-marked autophagosomes and, due to the greater resistance of mCherry than GFP to lysosomal proteases, the successful delivery of autophagosomes to the lysosome by mCherry-labeled autolysosomes (Kimura et al., 2007). In control IOMs, there were dual mCherry-GFP-positive autophagosomes, as well as just mCherry-positive degradative autolysosomes (Figure 4A). In contrast, the Rab2, Rab7 and Stx17 RNAi IOMs contained a striking increase in dual mCherry-GFP-positive puncta (Figure 4A–B), showing that autophagosome clearance was severely blocked either due to accumulation of autophagosomes or nondegradative autolysosomes. The Stx17 SNARE localizes to the outer membrane of fully formed autophagosomes then detaches upon lysosomal degradation of the autophagosomal inner membrane (Itakura et al., 2012; Tsuboyama et al., 2016). In the RNAi conditions, Stx17 localized to the vesicle membranes (Figure 4C) also marked with Atg8 (Figure 4D; 0.53 Pearson correlation), indicating their identity as primarily mature autophagosomes. Confirming these results, TEM myofiber transverse sections (Figure 2B) revealed Rab2, Rab7 or Stx17 RNAi-depleted IOMs similarly and uniformly filled with thousands of accumulated autophagosomes carrying nondegraded cytoplasm and organelles (Figure 4E–F). Thus, similar to previous reports of Rab7, Stx17, SNAP29 and Vamp7/8 functions in other Drosophila tissues (Hegedűs et al., 2016; Takáts et al., 2013), with myofiber remodeling, Rab2 also is required for autophagosome-lysosome fusion. Figure 4 Download asset Open asset Autophagosomes accumulate in IOMs with Rab2, Rab7 or Stx17 knockdown. All IOMs imaged at 4d APF. (A) Autophagic flux assay using tandem-tagged mCherry:GFP:Atg8 (mCherry (C), pink; GFP (G), green; colocalization, white). Peripheral IOM z-sections with magnified regions from indicated boxed areas shown below. In control IOMs, mCherry-positive only puncta were primarily detected, indicative of Atg8 flux to autolysosomes. In Rab2, Rab7 or Stx17 RNAi IOMs, dual-positive Atg8 puncta were primarily detected, indicating block in autophagic flux. (B) Pearson correlation between GFP and mCherry of mCherry:GFP:Atg8 from pooled data for 10 images from three experiments, ± SD. (C) GFP:Stx17 distribution in IOMs from control or with Rab2, Rab7 or SNAP29 RNAi, which show GFP:Stx17 at puncta and small (D) Colocalization of and in Rab7 RNAi IOMs. TEM images of IOM IOMs show myofibrils and T-tubule membranes, while Rab2, Rab7 or Stx17 RNAi IOMs were filled mostly with autophagosomes. (F) Quantification of the of autophagosomes IOM ± SD. Figure data 1 Relates Figure Pearson correlation indicating colocalization between GFP and mCherry from mCherry:GFP:Atg8 expressed in IOMs at 4d APF of control and RNAi conditions shown (.xlsx file). Download Figure data 2 Relates to both Figure and Figure Quantification of the of autophagosomes (Figure or (Figure IOM at 4d APF for the control and RNAi conditions shown (.xlsx file). Download We a requirement for autophagy in T-tubule remodeling by early steps in autophagy with muscle-targeted or In each RNAi resulted in disorganized and T-tubules and myofibrils in IOMs at 4d APF (Figure Furthermore, TEM analysis of IOMs with or RNAi showed a lack of T-tubules, disorganized myofibrils and a striking accumulation of that filled the (Figure The accumulation of (Figure confirmed the TEM of within the cytoplasm with RNAi (Figure or mature but blocked autophagosomes with Rab2 RNAi (Figure suggesting that are a cargo of autophagy during IOM remodeling. As for a block in autophagy and the Rab2 RNAi, autophagosomes not or accumulate in IOMs with and Figure 5 Download asset Open asset is required for IOM T-tubule remodeling and clearance. (A) mCD8:GFP in 4d APF dorsal abdominal muscles (top) and IOM (bottom) for control and or RNAi conditions. (B) T-tubule (Dlg1, green) and myofibril (F-actin, pink) organization in IOMs of control and or RNAi that show and disorganized (C) TEM images of IOM show disorganized contractile system, lack of T-tubules and many in or RNAi conditions at 4d APF. Quantification of the of ± SD. (E) green) and myofibril (F-actin, pink) organization in control and or Rab2 RNAi IOMs at 4d APF. is upregulated with a requirement for T-tubule membrane disassembly The autophagy requirement for T-tubule remodeling the questions of autophagy and required at distinct stages over IOM remodeling. In wildtype we used imaging to monitor the of autophagosomes and in live IOMs over 24h intervals of metamorphosis (Figure The of Atg8 puncta indicative of autophagosomes by 1d APF and then by 3d APF (Figure top and We confirmed a similar autophagosome distribution detected by of Atg8 in wildtype IOMs at 1d APF (Figure supplement These results the of uniformly mCD8:GFP-marked vesicles (Figure and autophagosomes detected by TEM (Figure over APF during wildtype IOM remodeling. the of and Rab7 marked or also during pupal stages APF (Figure the increase in autophagy at 1d APF not an autophagy role in myofiber cell as the IOMs throughout metamorphosis and days later with adult eclosion (Ribeiro et al., 2011). Figure with 2 see all Download asset Open asset is with and required for T-tubule membrane (A) Time course microscopy of in live wildtype animals over 1 intervals during (B) Quantification of puncta ± from at least 10 selected IOMs. (C–D) Time course microscopy of and in live wildtype animals at the indicated APF IOM by T-tubules ± and of autophagosomes ± quantified from at least 10 selected IOMs. (E) Time course microscopy during metamorphosis of mCD8:GFP in IOMs of Rab2 RNAi or RNAi in Figure 1F, Rab2 and RNAi show normal T-tubules in 3IL muscle and initial defects in membrane organization by 1d APF that persist as a block in remodeling through 4d APF. (F) mCD8:GFP membrane in central regions of IOMs at APF upon T-tubule disassembly in Rab2 RNAi or RNAi, with mCD8:GFP membrane reorganization into membrane or membrane vesicles at 1d APF in control and Rab2 RNAi IOMs. See Figure 1 and 2 for related data. Figure data 1 Relates to Figure Quantification of the of puncta IOM over the indicated from 3IL through 4d APF in wildtype myofibers (.xlsx file). Download Figure data 2 Relates to Figure Quantification of the of puncta IOM over the indicated from 3IL through 24h APF in wildtype myofibers (.xlsx file). Download Figure data 3 Relates to Figure Quantification of the IOM by for T-tubules over the indicated from 3IL through 24h APF in wildtype myofibers (.xlsx file). Download The autophagy APF (Figure to with the of T-tubule membranes (Figure To the timing and between autophagy and T-tubule we performed a more IOM remodeling between late larval to 1d APF stages. We quantified a of autophagy between and APF that by APF in IOMs (Figure top and In experiments, we used live imaging of a to T-tubules and the plasma While the T-tubules intact from third instar larval through APF stages, the T-tubule membrane network partially by APF and was completely absent by

  PublisherDOI

• VAMP8-3xHA Uptake Assay in HeLa Cells

  BIO-PROTOCOL · 2016-01-01 · 4 citations

  articleSenior author

  Transmembrane proteins are rarely exclusively localized to a specific vesicle or an organelle. Most transmembrane proteins undergo complicated trafficking routes. Thus, transmembrane proteins are under constant flux, and at steady state, found on a variety of vesicles or organelles. This characteristic makes the study of their trafficking routes complex, since at any given moment, different molecules are often being trafficked in opposing directions. Pulse-chase experiments can temporally track a specific pool of a transmembrane protein of interest, allowing for the kinetic description of its trafficking route. This type of technique has been used extensively to follow a large array of plasma membrane localized proteins (Diril et al., 2006; Jean et al., 2010). Here, we describe a method that allows the study of VAMP8 trafficking from the plasma membrane to endolysosomal compartments. This method was used to describe a role for MTMR13 and RAB21 in the regulation of VAMP8 trafficking to endolysosomes (Jean et al., 2015).

  PublisherDOI

• Mechanisms of muscle cell remodeling in Drosophila

  The Japanese Biochemical Society/The Molecular Biology Society of Japan · 2015-11-02

  articleSenior author
  Publisher

• Starvation‐induced MTMR13 and RAB21 activity regulates VAMP8 to promote autophagosome–lysosome fusion

  EMBO Reports · 2015-02-03 · 90 citations

  articleOpen accessSenior author
  PublisherOA PDFDOI

• Classes of phosphoinositide 3-kinases at a glance

  Journal of Cell Science · 2014-02-28 · 370 citations

  reviewOpen accessSenior author

  The phosphoinositide 3-kinase (PI3K) family is important to nearly all aspects of cell and tissue biology and central to human cancer, diabetes and aging. PI3Ks are spatially regulated and multifunctional, and together, act at nearly all membranes in the cell to regulate a wide range of signaling, membrane trafficking and metabolic processes. There is a broadening recognition of the importance of distinct roles for each of the three different PI3K classes (I, II and III), as well as for the different isoforms within each class. Ongoing issues include the need for a better understanding of the in vivo complexity of PI3K regulation and cellular functions. This Cell Science at a Glance article and the accompanying poster summarize the biochemical activities, cellular roles and functional requirements for the three classes of PI3Ks. In doing so, we aim to provide an overview of the parallels, the key differences and crucial interplays between the regulation and roles of the three PI3K classes.

  PublisherOA PDFDOI

Recent grants

• NIH Grant R01GM078176

  NIH · $1.6M · 2013

• T-tubule membrane remodeling in Drosophila myofiber function and models of myopathy

  NIH · $2.4M · 2018–2030

• NIH Grant P50GM085764

  NIH · $46.6M · 2019

Frequent coauthors

• Norbert Perrimon

  Howard Hughes Medical Institute

  25 shared

• Michael Boutros

  Medizinische Fakultät Mannheim

  12 shared

• Susan Armknecht

  Brown University

  11 shared

• Heidelberg Fly Array Consortium

  Harvard University

  9 shared

• Steve Jean

  Université de Sherbrooke

  9 shared

• Britta Koch

  University of Nottingham

  9 shared

• Renato Paro

  University of Basel

  9 shared

• Marc Hild

  9 shared

Awards & honors

• Howard Hughes Medical Institute Predoctoral Fellowship
• Fellow of the Jane Coffin Childs Memorial Fund for Medical R…
• Fellowship from The David & Lucille Packard Foundation
• Fellowship from The Sidney Kimmel Foundation for Cancer Rese…