Biology subjects

Lange, J. J.

Publications and source records attributed to Lange, J. J..

26 records · Page 2Linked to original sources

bioRxiv2021

Diverse mating phenotypes impact the spread of wtf meiotic drivers in S. pombe

Meiotic drivers are genetic loci that break Mendels law of segregation to be transmitted into more than half of the offspring produced by a heterozygote. The success of a driver relies on outcrossing because drivers gain their advantage in heterozygotes. It is, therefore, curious that Schizosaccharomyces pombe, a species reported to rarely outcross, harbors many meiotic drivers. To address this paradox, we measured mating phenotypes in S. pombe natural isolates. We found that the propensity to inbreed varies between natural isolates and can be affected both by cell density and by the available sexual partners. Additionally, we found that the observed level of inbreeding slows, but does not prevent, the spread of a wtf meiotic driver in the absence of additional fitness costs. These analyses reveal parameters critical to understanding the evolution of S. pombe and help explain the success of meiotic drivers in this species.

evolutionary biology↗

bioRxiv2021

Hox genes regulate asexual reproductive behavior and tissue segmentation in adult animals

Hox genes are highly conserved transcription factors renowned for their roles in the segmental patterning of the embryonic anterior-posterior (A/P) axis1. Emerging evidence for Hox gene expression and function in postnatally derived structures has fueled interest in their additional roles beyond embryogenesis2,3. We report novel functions for Hox genes in A/P adult tissue segmentation and transverse fission behavior underlying asexual reproduction in the planarian flatworm, Schmidtea mediterranea. Silencing of each of the planarian Hox family members identified 5 Hox genes required for asexual reproduction. Among these, silencing of hox3 genes resulted in supernumerary segments, while silencing of post2b eliminated segmentation altogether. The opposing roles of hox3 and post2b in segmentation are paralleled in their respective regulation of fission behavior. Silencing of hox3 increased the frequency of fission behavior initiation, while silencing of post2b eliminated fission behavior entirely. Furthermore, we identified a network of downstream effector genes mediating Hox gene regulation of asexual reproduction, thereby providing insight into their respective mechanisms of action. Our study establishes postembryonic roles for Hox genes in regulating the emergence of tissue segmentation and specific behaviors associated with asexual reproduction in adult animals.

developmental biology↗

bioRxiv2020

Mechanistic inferences from analysis of measurements of protein phase transitions in live cells

The combination of phase separation and disorder-to-order transitions can give rise to ordered, semi-crystalline fibrillar assemblies that underlie prion phenomena namely, the non-Mendelian transfer of information across cells. Recently, a method known as Distributed Amphifluoric Forster Resonance Energy Transfer (DAmFRET) was developed to study the convolution of phase separation and disorder-to-order transitions in live cells. In this assay, a protein of interest is expressed to a broad range of concentrations and the acquisition of local density and order, measured by changes in FRET, is used to map phase transitions for different proteins. The high-throughput nature of this assay affords the promise of uncovering sequence-to-phase behavior relationships in live cells. Here, we report the development of a supervised method to obtain automated and accurate classifications of phase transitions quantified using the DAmFRET assay. Systems that we classify as undergoing two-state discontinuous transitions are consistent with prion-like behaviors, although the converse is not always true. We uncover well-established and surprising new sequence features that contribute to two-state phase behavior of prion-like domains. Additionally, our method enables quantitative, comparative assessments of sequence-specific driving forces for phase transitions in live cells. Finally, we demonstrate that a modest augmentation of DAmFRET measurements, specifically time-dependent protein expression profiles, can allow one to apply classical nucleation theory to extract sequence-specific lower bounds on the probability of nucleating ordered assemblies. Taken together, our approaches lead to a useful analysis pipeline that enables the extraction of mechanistic inferences regarding phase transitions in live cells.

biophysics↗

bioRxiv2020

Driving Integrative Structural Modeling with Serial Capture Affinity Purification

Streamlined characterization of protein complexes remains a challenge for the study of protein interaction networks. Here, we describe Serial Capture Affinity Purification (SCAP) where two separate proteins are tagged with either the HaloTag or the SNAP-tag, permitting a multi-step affinity enrichment of specific protein complexes. The multifunctional capabilities of these protein tagging systems also permit in vivo validation of interactions using FRET and FCCS quantitative imaging. When coupling SCAP to cross-linking mass spectrometry, an integrated structural model of the complex of interest can be generated. We demonstrate this approach using the Spindlin1 and SPINDOC chromatin associated protein complex, culminating in a structural model with two SPINDOC docked on one SPIN1 molecule. In this model, SPINDOC interacts with the SPIN1 interface previously shown to bind a lysine and arginine methylated sequence of histone H3 Taken together, we present an integrated affinity purification, live cell imaging, and cross linking mass spectrometry approach for the building of integrative structural models of protein complexes.

systems biology↗

bioRxiv2020

The wtf4 meiotic driver utilizes controlled protein aggregation to generate selective cell death

Meiotic drivers are parasitic loci that force their own transmission into greater than half of the offspring of a heterozygote. Many drivers have been identified, but their molecular mechanisms are largely unknown. The wtf4 gene is a meiotic driver in Schizosaccharomyces pombe that uses a poison-antidote mechanism. Here, we show that the Wtf4 proteins can function outside of gametogenesis and in a distantly related species, Saccharomyces cerevisiae. The Wtf4poison protein forms dispersed, toxic aggregates. The similar Wtf4antidote protein also forms aggregates but is sequestered within or near vacuoles and is mostly benign. The Wtf4antidote can co-assemble with the Wtf4poison and promote its trafficking to vacuoles. We show that neutralization of the Wtf4poison requires both co-assembly with the Wtf4antidote and aggregate sequestration, as mutations that disrupt either of these processes results in cell death. This work reveals that wtf parasites can exploit protein aggregate management pathways to selectively destroy gametes.

cell biology↗

bioRxiv2019

Dramatically diverse S. pombe wtf meiotic drivers all display high gamete-killing efficiency

Meiotic drivers are selfish genetic loci that force their transmission into more than 50% of the viable gametes made by heterozygotes. Meiotic drivers are known to cause infertility in a diverse range of eukaryotes and are predicted to affect the evolution of genome structure and meiosis. The wtf gene family of Schizosaccharomyces pombe includes both meiotic drivers and drive suppressors and thus offers a tractable model organism to study drive systems. Currently, only a handful of wtf genes have been functionally characterized and those genes only partially reflect the diversity of the wtf gene family. In this work, we functionally test 22 additional wtf genes. We identify eight new drivers that share between 30-90% amino acid identity with previously characterized drivers. Despite the vast divergence between these genes, they generally drive into >85% gametes when heterozygous. We also find three new wtf genes that suppress drive, including two that also act as autonomous drivers. Additionally, we find that wtf genes do not underlie a weak (64%) transmission bias caused by a locus or loci on chromosome 1. Finally, we find that some Wtf proteins have expression or localization patterns that are distinct from the poison and antidote proteins encoded by drivers and suppressors, suggesting some wtf genes may have non-meiotic drive functions. Overall, this work expands our understanding of the wtf gene family and the burden selfish driver genes impose on S. pombe.\n\nArticle SummaryDuring gametogenesis, the two gene copies at a given locus, known as alleles, are each transmitted to 50% of the gametes (e.g. sperm). However, some alleles cheat so that they are found in more than the expected 50% of gametes, often at the expense of fertility. This selfish behavior is known as meiotic drive. Some members of the wtf gene family in the fission yeast, Schizosaccharomyces pombe, kill the gametes (spores) that do not inherit them, resulting in meiotic drive favoring the wtf allele. Other wtf genes act as suppressors of drive. However, the wtf gene family is diverse and only a small subset of the genes has been characterized. Here we analyze the functions of other members of this gene family and found eight new drivers as well as three new suppressors of drive. Surprisingly, we find that drive is relatively insensitive to changes in wtf gene sequence as highly diverged wtf genes execute gamete killing with similar efficiency. Finally, we also find that the expression and localization of some Wtf proteins are distinct from those of known drivers and suppressors, suggesting that these proteins may have non-meiotic drive functions.

genetics↗

bioRxiv2017

Quinary structure kinetically controls protein function and dysfunction

Protein self-assemblies modulate protein activities over biological time scales that can exceed the lifetimes of the proteins or even the cells that harbor them. We hypothesized that these time scales relate to kinetic barriers inherent to the nucleation of ordered phases. To investigate nucleation barriers in living cells, we developed Distributed Amphifluoric FRET (DAmFRET). DAmFRET exploits a photoconvertible fluorophore, heterogeneous expression, and large cell numbers to quantify via flow cytometry the extent of a proteins self-assembly as a function of cellular concentration. We show that kinetic barriers limit the nucleation of ordered self-assemblies, and that the persistence of the barriers with respect to concentration relates to structure. Supersaturation resulting from sequence-encoded nucleation barriers gave rise to prion behavior, and enabled a prion-forming protein, Sup35 PrD, to partition into dynamic intracellular condensates or to form toxic aggregates. Our results suggest that nucleation barriers govern cytoplasmic inheritance, subcellular organization, and proteotoxicity.\n\nHighlightsO_LIDistributed Amphifluoric FRET (DAmFRET) quantifies nucleation in living cells\nC_LIO_LIDAmFRET rapidly distinguishes prion-like from non-prion phase transitions\nC_LIO_LINucleation barriers allow switch-like temporal control of protein activity\nC_LIO_LISequence-intrinsic features determine the concentration-dependence of nucleation barriers\nC_LI

biophysics↗

bioRxiv2017

NECAPs are negative regulators of the AP2 clathrin adaptor complex

Eukaryotic cells internalize transmembrane receptors via clathrin-mediated endocytosis, but it remains unclear how the machinery underpinning this process is regulated. We recently discovered that membrane-associated muniscin proteins such as FCHo and SGIP initiate endocytosis by converting the AP2 clathrin adaptor complex to an open, active conformation that is then phosphorylated (Hollopeter et al., 2014). Here we report that loss of ncap-1, the sole C. elegans gene encoding an adaptiN Ear-binding Coat-Associated Protein (NECAP), bypasses the requirement for FCHO-1. Biochemical analyses reveal AP2 accumulates in an open, phosphorylated state in ncap-1 mutant worms, suggesting NECAPs promote the closed, inactive conformation of AP2. Consistent with this model, NECAPs preferentially bind open and phosphorylated forms of AP2 in vitro and localize with constitutively open AP2 mutants in vivo. NECAPs do not associate with phosphorylation-defective AP2 mutants, implying that phosphorylation precedes NECAP recruitment. We propose NECAPs function late in endocytosis to inactivate AP2.

cell biology↗