Biology subjects

Affolter, M.

Publications and source records attributed to Affolter, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Tag-stabilized fluorescent protein binders for endogenous cell type-specific protein labelling

Protein labelling using genetically encoded fluorescent protein fusions has revolutionized the study of protein localization and function. However, visualizing endogenous proteins in specific cell types remains challenging, as existing approaches often require overexpression or laborious genetic engineering. To address these limitations, we developed a library of fluorescent protein binders using existing nanobodies and single-chain variable fragments that recognize fluorescent proteins and short peptide tags, allowing for modular, cell type-specific labelling of genetically tagged endogenous proteins. We found that fluorescent protein binders generate background fluorescence in the absence of the target protein, reducing signal specificity. To overcome this limitation, we generated destabilized fluorescent protein binders directed against GFP, ALFA, GCN4 peptide and V5. These binders display increased antigen-dependent stability and reduced background fluorescence, thereby improving the signal-to-noise ratio of endogenous protein visualization. We demonstrate the detection of endogenous proteins in drosophila larvae and embryos, zebrafish embryos, as well as in specific neuronal populations of the adult drosophila brain. In the brain, tissue-specific labelling revealed previously unknown protein expression and subcellular localization in sleep-regulatory neurons. Together, our panel of tag-stabilized fluorescent protein binders provides a versatile, ready-to-use toolkit for background-free, cell type-specific visualization of endogenous proteins.

cell biology↗

bioRxiv2025

Genetic characterization of the apterous Life Span Enhancer in Drosophila melanogaster

The selector gene apterous (ap) of Drosophila melanogaster is best known for its prominent role in wing formation. apnull flies are viable but have no wings. However, apnull flies are associated with another phenotype whose analysis was largely neglected in the past 40 years: adults die precociously 2-3 days after eclosion. We have recently published a comprehensive analysis on the physiology of this striking phenotype. We showed that Ap protein is essential early in metamorphosis during hindgut remodeling where it is required for rectal papillae formation in the rectal ampulla. Wild-type flies have four rectal papillae that are involved in selective reabsorption of essential ions and metabolites from the primary urine produced by the Malpighian tubules. In apnull flies, rectal papillae formation is aborted prematurely. The corresponding papillar cells remain stuck in the rectal lumen. This leads to complete intestinal blockage and a cascade of pathologies resulting in premature death. In this study, we genetically identify and functionally dissect a novel tissue specific cis- regulatory element that directs Ap expression in the hindgut. This Life Span Enhancer (apLSE) maps to a [~]400 bp DNA fragment that is activated via only two small regulatory modules. Genetic analysis of apLSE alleles proves that they are essential for papilla formation and thus for adult survival. Our studies solve the 110-year-old enigma about the ap syndrome. They establish the apLSE as a key player linking hindgut morphogenesis and survival. We propose that several aspects of the ap syndrome are a consequence of drastic changes in endocrine homeostasis.

developmental biology↗

bioRxiv2025

An AmotL2 - Yap1 Module Integrates Flow and Junctional Mechanics to Specify Vascular Pruning Hotspots

Vascular networks constantly remodel to optimise blood flow, yet how endothelial cells (ECs) integrate haemodynamic cues with junctional mechanics to execute precise branch pruning remains unclear. Using live imaging and quantitative topology analysis in the zebrafish sub-intestinal vein plexus, we define the cellular events underlying flow-guided regression. Pruning occurs in geometrically disadvantaged, low-flow branches and is executed through transient polarity fluctuations, junctional remodelling and directed EC migration. We identify AmotL2 as an essential regulator of endothelial mechanical competence. AmotL2 maintains junctional architecture, actomyosin tension and appropriate cell packing, enabling ECs to polarise and rearrange in response to local flow differences. Loss of AmotL2 disrupts tension homeostasis, increases tissue compaction and prevents branch regression despite normal perfusion. In contrast, Yap1 plays an opposing, stabilising role, and its loss results in excessive pruning and network simplification. Together, AmotL2 and Yap1 constitute a mechanosensitive balancing module that interprets flow magnitude and vascular geometry to determine branch stability. This framework describes how mechanical forces sculpt vascular architecture during development and homeostasis.

developmental biology↗

bioRxiv2025

Direct cell-to-cell transport of Hedgehog morphogen is aided by the diffusible carrier Shifted/DmWif1

The establishment of morphogen gradients is fundamental during embryonic patterning. Yet, the mechanisms by which morphogens disperse remain highly controversial. In a paradigmatic example, the dispersal of the morphogen Hedgehog (Hh) is both limited by and dependent on its lipid modifications. To address this paradox, several competing transport models have been proposed, ranging from lipid-shielding solubilizing factors to transport via specialized filopodia called cytonemes. In this study, we uncover that the lipid-shielding protein DmWif1/Shifted (Shf) participates in the contact-dependent cell transfer of Hh. Using nanobody-based immobilization, we demonstrate the tight association of Shf and Hh to cytoneme membranes. We show that Shf is anchored to membranes by the Hh co-receptor Interference Hedgehog (Ihog) and that the Ihog-Shf complex is required for Hh dispersal and signaling. Together, our results unify cytoneme-mediated transport and membrane release into a single dispersal model, in which local solubilization is required for cytonemes to exchange Hh-bound Shf at cell-cell contacts between presenting and receiving cytonemes.

developmental biology↗

bioRxiv2025

Intestinal control of feeding initiation in Drosophila melanogaster

The interplay between feeding and excretion is essential for organismal nutrition and survival, yet their mechanistic coupling remains poorly understood. At the onset of life, feeding must be initiated while developmental waste products - the meconium - need to be eliminated. Using Drosophila as a model system, we explored the in vivo mechanisms coordinating these processes. We developed novel behavioral assays for newly eclosed flies and discovered that, similar to neonatal mammals, Drosophila excrete their meconium shortly after eclosion. Remarkably, feeding initiation occurs only after partial meconium elimination. We identified a cis-regulatory element associated with the apterous gene, which, when disrupted, prevents both meconium excretion and adult feeding initiation. These flies develop hindgut obstruction (ileus), avoid food, and exhibit increased proboscis extension sleep - a behaviour we found plays a functional role in waste clearance under normal conditions. Through experimental inhibition of meconium excretion, we established that this process is prerequisite for feeding initiation, suggesting a gut-to-brain signaling circuit that couples these fundamental physiological processes. The progression of phenotypes we observed parallels the hallmarks of mechanical ileus in humans. Our findings reveal previously unrecognized links between intestinal clearance, feeding behavior, and survival, with potential implications for understanding similar processes across species.

developmental biology↗

bioRxiv2024

In vivo regulation of an endogenously-tagged protein by a light-regulated kinase

Post-translational modifications (PTMs) are indispensable modulators of protein activity. Most cellular behaviours, from cell division to cytoskeletal organization, are controlled by PTMs, their miss-regulation being associated with a plethora of human diseases. Traditionally, the role of PTMs has been studied employing biochemical techniques. However, these approaches fall short when studying PTM dynamics in vivo. In recent years, functionalized protein binders have allowed the post-translational modification of endogenous proteins by bringing an enzymatic domain in close proximity to the protein they recognize. To date, most of these methods lack the temporal control necessary to understand the complex effects triggered by PTMs. In this study, we have developed a method to phosphorylate endogenous Myosin in a light-inducible manner. The method relies both on nanobody-targeting and light-inducible activation in order to achieve both tight specificity and temporal control. We demonstrate that this technology is able to disrupt cytoskeletal dynamics during Drosophila embryonic development. Together, our results highlight the potential of combining optogenetics and protein binders for the study of the proteome in multicellular systems.

developmental biology↗

bioRxiv2024

Initiation of Lumen Formation from Junctions via Differential Actomyosin Contractility Regulated by Dynamic Recruitment of Rasip1

Vascular lumens are essential for the dispersal of nutrients, oxygen, and immune cells throughout the body. De novo lumen formation necessitates the precise segregation of junctional proteins from apical surfaces. Direct visualization of this process and a comprehensive understanding on how the apical and junctional compartments are segregated and established remain elusive, due to the lack of visualizable systems, proper reporters and manipulative tools. Using zebrafish as an experimental model, we have established a wealth of molecular reporters, photo-convertible and optogenetic tools and combined them with genetic analyses to study the establishment of apical domains from pre-apical junctional complexes. Our study identified Rasip1 as one of the earliest apical proteins recruited, peripheralizing cortical contractility at the junctional patches by inhibiting NMII at the center, thereby initiating the outward flow of junctional complexes. After the establishment of the apical compartments, Rasip1 shuttles between junctions and the apical compartments to the high-tension sites and tunes down the local hyper contractility, restricting Cdh5 to junctions by suppressing apical contractility. Above all, the formation of de novo lumens and maintenance of junctional integrity depend on the precise control of contractility within the apical compartments and junctions, orchestrated by the dynamic recruitment of Rasip1.

developmental biology↗

bioRxiv2023

Transcriptional control of compartmental boundary positioning during Drosophila wing development

The establishment of tissue axes is fundamental during embryonic development. In the Drosophila wing, the anterior/posterior (AP) and the dorsal/ventral (DV) compartment boundaries provide the basic coordinates around which the tissue develops. These boundaries arise as a result of two lineage decisions, the acquisition of posterior fate by the selector gene engrailed (en) and dorsal fate by the selector gene apterous (ap). While the en expression domain is set up during embryogenesis, ap expression begins only during early wing development. Thus, the correct establishment of the ap expression pattern relative to en must be tightly controlled. Here, we functionally investigate the transcriptional inputs integrated by the early ap enhancer (apE) and their requirement for correct boundary formation. Detailed mutational analyses using CRISPR/Cas revealed a role for apE in positioning the DV boundary relative to the AP boundary, with apE mutants often displaying mirror-image anterior wing duplications. We then designed and applied methods to accomplish tissue-specific enhancer disruption via dCas9 expression. This approach allowed us to dissect the spatiotemporal requirement for apE function, clarifying the mechanism by which apE misregulation leads to AP defects. Base-pair-resolution analyses of apE uncovered a single HOX binding site essential for wing development that, when mutated, led to wingless flies. We demonstrated that the transcription factors Pointed (Pnt), Homothorax (Hth), and Grain (Grn) are required for apE function, and the HOX gene Antennapedia (Antp) contributes to early wing development. Together, our results provide a comprehensive molecular basis of early ap activation and the developmental consequences of its misregulation, shedding light on how compartmental boundaries are set up during development.

developmental biology↗

bioRxiv2023

Genetic analysis of rab7 mutants in zebrafish

Vascular network formation requires the fusion of newly formed blood vessels and the emergence of a patent lumen between the newly established connections so that blood flow can start. Lumen formation has been shown to depend on the late endosomal/lysosomal pathway in various organs of animal tubular systems. Here, we identified a late endosomal/lysosomal vesicular fraction (Rab7/Lamp2) in early zebrafish angiogenic sprouts, which appears to contribute to apical membrane growth during lumen formation. To study the effect of the late endocytic pathway on vascular development, we generated mutant alleles for all three rab7 genes in zebrafish (rab7a, rab7ba, rab7bb). All rab7 genes are expressed in wild-type zebrafish and we did not detect any compensatory effects by the other rab7 isoforms in single knockout mutants, which were all viable. Only the triple mutant was lethal suggesting some functional redundancy. However, the different rab7 isoforms fulfil also at least partially independent functions because eggs laid from mothers lacking two rab7 (rab7a and/or rab7bb). showed reduced survival and contained enlarged yolk granules, suggesting maternal contribution of these two rab7. Finally, we observed minor effects on lumen formation in embryos which still express one copy of rab7. Our results support the notion that the late endocytic/lysosomal compartment contributes to lumen expansion.

genetics↗

bioRxiv2022

Next-generation plasmids for transgenesis in zebrafish and beyond

Transgenesis is an essential technique for any genetic model. Tol2-based transgenesis paired with Gateway-compatible vector collections has transformed zebrafish transgenesis with an accessible, modular system. Here, we established several next-generation transgenesis tools for zebrafish and other species to expand and enhance transgenic applications. To facilitate gene-regulatory element testing, we generated Gateway middle entry vectors harboring the small mouse beta-globin minimal promoter coupled to several fluorophores, CreERT2, and Gal4. To extend the color spectrum for transgenic applications, we established middle entry vectors encoding the bright, blue-fluorescent protein mCerulean and mApple as an alternative red fluorophore. We present a series of p2A peptide-based 3 vectors with different fluorophores and subcellular localizations to co-label cells expressing proteins of interest. Lastly, we established Tol2 destination vectors carrying the zebrafish exorh promoter driving different fluorophores as a pineal gland-specific transgenesis marker active prior to hatching and through adulthood. exorh-based reporters and transgenesis markers also drive specific pineal gland expression in the eye-less cavefish (Astyanax). Together, our vectors provide versatile reagents for transgenesis applications in zebrafish, cavefish, and other models.

genetics↗

bioRxiv2022

Heterodimerization-dependent secretion of BMPs in Drosophila

Combinatorial signaling is key to instruct context-dependent cell behaviors. During embryonic development, adult homeostasis and disease, Bone Morphogenetic Proteins (BMPs) act as dimers to instruct specific cellular responses. BMP ligands can form both homo- or heterodimers; however, obtaining direct evidence of the endogenous localization and function of each form has proven challenging. Here, we make use of precise genome editing and direct protein manipulation via protein binders to dissect the existence and functional relevance of BMP homo- and heterodimers in the Drosophila wing imaginal disc. This approach revealed in situ the existence of Dpp (BMP2/4)/Gbb (BMP5/6/7/8) heterodimers. We found that Gbb, despite being produced by all the cells of the wing imaginal disc, is only secreted by the cells in which Dpp is expressed. Dpp and Gbb form a gradient of heterodimers whereas neither Dpp nor Gbb homodimers are evident under endogenous physiological conditions. We find that the formation of heterodimers is critical for obtaining optimal signaling and long-range BMP distribution in the developing wing. These results indicate that Dpp/Gbb heterodimers are the active signal required for epithelial patterning and growth.

developmental biology↗

bioRxiv2022

In vivo seamless genetic engineering via CRISPR-triggered single-strand annealing

Precise genome engineering is essential for both basic and applied research. CRISPR/Cas accelerated the speed and ease by which defined exogenous sequences are integrated into specific loci. Nevertheless, knock-in generation in multicellular animals remains challenging, partially due to the complexity of insertion screening. Even when achieved, the analysis of protein localization can still be unfeasible in highly packed tissues, where spatial and temporal control of gene labeling would be ideal. Here, we describe SEED/Harvest, an efficient knock-in method based on homology-directed (HDR) and single-strand annealing (SSA) repair pathways. HDR mediates the integration of a switchable cassette. Upon a subsequent CRISPR-triggered repair event, resolved by SSA, the cassette is seamlessly removed. Germline excision of SEED cassettes allows for fast and robust knock-in generation with both fluorescent proteins and short protein tags in tandem. Tissue-specific expression of Cas9 results in somatic cassette excision, conferring spatio-temporal control of protein labelling and the conditional rescue of mutants. Finally, to achieve conditional protein labeling and manipulation of short tag knock-ins, we have developed a toolbox based on rational engineering and functionalization of the ALFA nanobody.

genetics↗

bioRxiv2021

In vivo regulation of fluorescent fusion proteins by engineered kinases

Reversible protein phosphorylation by kinases in extensively used to control a plethora of processes essential for proper development and homeostasis of multicellular organisms. One main obstacle in studying the role of a defined kinase-substrate interaction is that kinases form complex signaling networks and most often phosphorylate multiple substrates involved in various cellular processes. In recent years, several new approaches have been developed to control the activity of a given kinase. However, most of them fail to regulate a single protein target, likely hiding the effect of a unique kinase-substrate by pleiotropic effects. To overcome this limitation, we have created protein binder-based engineered kinases for direct, robust and tissue-specific phosphorylation of target fluorescent protein fusions in vivo. We show that synthetic Rok kinases, based on the Drosophila ortholog of Rho-associated protein kinase (ROCK), are functional enzymes and can activate myosin II through phosphorylation of Sqh::GFP or Sqh::mCherry in different morphogenetic processes in a developing fly embryo. We next use the system to study the impact of actomyosin activation specifically in the developing tracheal branches and showed that ectopic activation of actomyosin with engineered Rok kinase did not prevent cell intercalation nor the formation of autocellular junctions. We assume that this approach can be adapted to other kinases and targets in various eukaryotic genetic systems.

developmental biology↗

bioRxiv2021

Vinculin controls endothelial cell junction dynamics during vascular lumen formation

Blood vessel morphogenesis is driven by coordinated endothelial cell behaviors, which depend on dynamic cell-cell interactions. Remodeling of endothelial cell-cell junctions promote morphogenetic cellular events while preserving vascular integrity. Here, we have analyzed the dynamics of endothelial cell-cell junctions during lumen formation in angiogenic sprouts. By live-imaging of the formation of intersegmental blood vessels in zebrafish, we demonstrate that lumen expansion is accompanied by the formation of transient finger-shaped junctions. Formation and maintenance of these junctional fingers are positively regulated by blood pressure whereas inhibition of blood flow prevents their formation. Using fluorescent reporters, we show that the tension-sensor Vinculin localizes to junctional fingers. Furthermore, loss of vinculin function, in vinculin a and -b double knockouts, prevents junctional finger formation in angiogenic sprouts, whereas endothelial expression of a vinculin transgene is sufficient to restore junctional fingers. Taken together, our findings suggest a mechanism in which lumen expansion during angiogenesis leads to an increase in junctional tension, which triggers recruitment of vinculin and formation of junctional fingers. We propose that endothelial cells may employ force-dependent junctional remodeling to react to changes in external forces to protect cell-cell contacts and to maintain vascular integrity during sprouting angiogenesis.

cell biology↗

bioRxiv2020

Asymmetric requirement of Dpp/BMP morphogen dispersal in the Drosophila wing disc

How morphogen gradients control patterning and growth in developing tissues remains largely unknown due to lack of tools manipulating morphogen gradients. Here, we generate two membrane-tethered protein binders that manipulate different aspects of Decapentaplegic (Dpp), a morphogen required for overall patterning and growth of the Drosophila wing. "HA trap" is based on a single-chain variable fragment (scFv) against the HA tag that traps HA-Dpp to mainly block its dispersal, while "Dpp trap" is based on a Designed Ankyrin Repeat Protein (DARPin) against Dpp that traps Dpp to block both its dispersal and signaling. Using these tools, we found that, while posterior patterning and growth require Dpp dispersal, anterior patterning and growth largely proceed without Dpp dispersal. We show that dpp transcriptional refinement from an initially uniform to a localized expression and persistent signaling in transient dpp source cells render the anterior compartment robust against the absence of Dpp dispersal. Furthermore, despite a critical requirement of dpp for the overall wing growth, neither Dpp dispersal nor direct signaling is critical for lateral wing growth after wing pouch specification. These results challenge the long-standing dogma that Dpp dispersal is strictly required to control and coordinate overall wing patterning and growth.

developmental biology↗

bioRxiv2020

Control of dynamic cell behaviors during angiogenesis and anastomosis by Rasip 1

Organ morphogenesis is driven by a wealth of tightly orchestrated cellular behaviors, which ensure proper organ assembly and function. Many of these cell activities involve cell-cell interactions and remodeling of the F-actin cytoskeleton. Here, we analyze the requirement for Rasip1 (Ras-interacting protein 1), an endothelial-specific regulator of junctional dynamics, during blood vessel formation. Phenotype analysis of rasip1 mutants in zebrafish embryos reveal distinct requirements for Rasip1 during sprouting angiogenesis, vascular anastomosis and lumen formation. During angiogenic sprouting, Rasip1 is required for efficient cell pairing, which is essential for multicellular tube formation. High-resolution time-lapse analyses show that these cell pairing defects are caused by a destabilization of tricellular junctions suggesting that tri-cellular junctions may serve as a counterfort to tether sprouting endothelial cells during morphogenetic cell rearrangements. During anastomosis, Rasip1 is required to establish a stable apical membrane compartment; rasip1 mutants display ectopic, reticulated junctions and the apical compartment is frequently collapsed. Loss of Ccm1 and Heg1 function leads to junctional defects similar to those seen in rasip1 mutants. Analysis of radil-b single and rasip1/radil-b double mutants reveal distinct and overlapping functions of both proteins. While Rasip1 and Radil-b have similar functions during angiogenic sprouting, the junction formation during anastomosis may primarily depend on Rasip1.

developmental biology↗

bioRxiv2020

A new perspective on the evolution of the interaction between the Vg/VGLL1-3 proteins and the TEAD transcription factors

The most downstream elements of the Hippo pathway, the TEAD transcription factors, are regulated by several cofactors, such as Vg/VGLL1-3. Earlier findings on human VGLL1 and here on human VGLL3 show that these proteins interact with TEAD via a conserved amino acid motif called the TONDU domain. Surprisingly, our studies reveal that the TEAD-binding domain of Drosophila Vg and of human VGLL2 is more complex and contains an additional structural element, an {Omega}-loop, that contributes to TEAD binding and in vivo function. To explain this unexpected structural difference between proteins from the same family, we propose that, after the genome-wide duplications at the origin of vertebrates, the {Omega}-loop present in an ancestral VGLL gene has been lost in some VGLL variants. These findings illustrate how structural and functional constraints can guide the evolution of transcriptional cofactors to preserve their ability to compete with other cofactors for binding to transcription factors.

biochemistry↗

bioRxiv2020

Protein manipulation using single copies of short peptide tags in cultured cells and in Drosophila melanogaster

Cellular development and specialized cellular functions are regulated processes which rely on highly dynamic molecular interactions among proteins, distributed in all cell compartments. Analysis of these interactions and their mechanisms of action has been one of the main topics in cellular and developmental research over the last fifty years. Studying and understanding the functions of proteins of interest (POIs) has been mostly achieved by their alteration at the genetic level and the analysis of the phenotypic changes generated by these alterations. Although genetic and reverse genetic technologies contributed to the vast majority of information and knowledge we have gathered so far, targeting specific interactions of POIs in a time- and space-controlled manner or analyzing the role of POIs in dynamic cellular processes such as cell migration or cell division would require more direct approaches. The recent development of specific protein binders, which can be expressed and function intracellularly, together with several improvements in synthetic biology techniques, have contributed to the creation of a new toolbox for direct protein manipulations. We selected a number of short tag epitopes for which protein binders from different scaffolds have been developed and tested whether these tags can be bound by the corresponding protein binders in living cells when they are inserted in a single copy in a POI. We indeed find that in all cases, a single copy of a short tag allows protein binding and manipulation. Using Drosophila, we also find that single short tags can be recognized and allow degradation and relocalization of POIs in vivo.

cell biology↗