Biology subjects

Michaeli, S.

Publications and source records attributed to Michaeli, S..

13 recordsLinked to original sources

bioRxiv2026

Evolution of a genome-architecture-encoded gene regulation system in trypanosomatids

Transcriptional regulation of protein-coding genes is a hallmark of eukaryotic gene expression. Yet, a group of parasitic protists, trypanosomatids, appear to lack this capability. Here, we analyzed genomic, nascent transcriptomic, RNA polymerase occupancy and gene organization data to reconstruct the evolutionary origin and biological consequences of their unusual regulatory strategy. Across 59 Discoba protists, we show stepwise evolutionary erosion of conventional transcription regulation components in trypanosomatida lineage, including gene consolidation into polycistronic transcription units (PTUs), shortening of intra-PTU non-coding regions, and depletion of transcription factors and their enriched DNA-binding motifs. This transition was associated with near-constitutive expression of most genes, indicating broad loss of conditional gene expression. However, trypanosomatids retain some differential regulation at the PTU level, with >70% PTUs featuring significantly different nascent transcription than their neighbors or resident chromosomes. Moreover, gene expression is not uniform within PTUs: nascent transcription, translation efficiency, and protein abundance progressively decline with distance from the transcription start site. Consistent with this architecture-encoded regulatory logic, co-complex subunits and co-pathway enzymes preferentially occupy adjacent positions within PTUs despite each PTUs overall functional heterogeneity. These findings reveal an evolutionary shift from gene-specific transcriptional regulation toward a regime where genome architecture becomes a regulator of gene expression.

evolutionary biology↗

bioRxiv2025

The evolutionary origin of host association and polycistronic transcription in trypanosomatids

Trypanosomatids (Kinetoplastids) encompass multiple lineages of parasitic protists with monoxenous or dixenous life cycles, infecting insects, vertebrates, and plants; in vertebrate hosts, some are intracellular, while others are extracellular. To understand the origin and diversification of their host associations, we integrated comparative genomics across 47 genomes. Results highlight that monoxenous, extracellular trypanosomatids originated from predatory ancestors through reductive evolution, which diminished their metabolic and hunting capabilities. Intracellularity and dixenous lifestyle convergently originated three times independently. Progressive consolidation of genes into polycistronic transcription units (PTUs) was a central innovation that began in early Glycomonada and expanded through chromosome fission-fusion and gene relocation/inversion. In present-day PTUs, protein complex subunits and metabolic pathway enzymes are positioned for co-expression in temporal synchrony, and chromosomes minimize colinear PTUs to counter transcriptional readthrough. Together, these results provide a time-resolved origin of host-association and polycistronic transcription in trypanosomatids, possibly through an intermediate phase of facultative parasitism.

evolutionary biology↗

bioRxiv2025

Mapping regulatory networks underlying Leishmania stage differentiation reveals an essential role for protein degradation in parasite development

Vector-borne, protist parasites have evolved complex developmental programs to adapt to very distinct host environments. How these important pathogens transition between insect and mammalian stages is only poorly understood. Here we investigate stage differentiation in the trypanosomatid parasite Leishmania that shows constitutive gene transcription, thus providing a unique model system to assess how development is governed by post-transcriptional mechanisms. Using a five-layer integrative systems analysis (from genome to metabolome), we examined hamster-isolated Leishmania donovani amastigotes and culture-derived, insect-stage promastigotes. This approach enabled us to rule out genomic adaptation as a key driver of parasite stage differentiation, while confirming the pivotal role of differential mRNA turnover in stage-specific gene expression. Assessing transcriptomic against proteomic expression changes uncovered an unexpectedly broad dynamic range of stage-regulated changes in protein abundance that only poorly correlated with mRNA levels. This discrepancy correlated with (i) alterations in snoRNA expression and changes in rRNA modification they guide suggesting stage-specific adaptation of the protein translation apparatus that can uncouple mRNA from protein abundancies, and (ii) differential protein degradation as revealed by quantitative proteomics of parasites treated with the proteasomal inhibitor lactacystin. Lactacystin treatment stalled the transition of spleen-derived amastigotes into promastigotes in culture, further underscoring the role of proteasomal activity in stage differentiation. Integration of our five-layer systems analysis established the first link between Leishmania development and the expression of co-regulated genetic networks encompassing mRNA turnover, protein translation, phosphorylation, and degradation. Our findings provide a powerful new resource for research programs that aim to dissect the emergent properties of regulatory networks and feedback loops underlying Leishmania stage differentiation, serving as a blueprint for other vector-borne pathogens that rely on disease-associated developmental transitions.

systems biology↗

bioRxiv2025

Simultaneous zero echo time fMRI of rat brain and spinal cord

PurposeFunctional assessments of the central nervous system (CNS) are essential for many areas of research. Functional MRI (fMRI) typically targets either the brain or the spinal cord, but usually not both, due to the obstacles associated with simultaneous image acquisitions from distant fields of view (FOVs) with conventional MRI. In this work, we establish a novel MRI approach that enables artefact-free, quiet, simultaneous fMRI of both brain and spinal cord, avoiding the need for dynamic shimming procedures. MethodsWe utilized zero echo time (TE) Multi-Band-SWeep Imaging with Fourier Transformation (MB-SWIFT) technique at 9.4T in a simultaneous dual-FOV configuration and two separate radio frequency (RF) transmit-receive surface coils. The first coil covered the rat brain, while the second was positioned approximately at the T13-L1 level of the rat"s spinal cord with copper shielding to minimize the coupling between the RF coils. Eight Sprague-Dawley rats were used for hindlimb stimulation fMRI studies. ResultsRobust and specific activations were detected in both the brain and spinal cord during hind paw stimulation at individual and group levels. The results established the feasibility of the novel approach for simultaneous functional assessment of the lumbar spinal cord and brain in rats. ConclusionThis study demonstrated the feasibility of a novel dual-FOV fMRI approach based on zero-TE MB-SWIFT and set the stage for translation to humans. The methodology enables comprehensive functional CNS evaluations of great value in different conditions such as pain, spinal cord injury, neurodegenerative diseases, and aging.

neuroscience↗

bioRxiv2024

Whisker stimulation with different frequencies reveals non-uniform modulation of functional magnetic resonance imaging signal across sensory systems in awake rats

Primary sensory systems are classically considered to be separate units, however there is current evidence that there are notable interactions between them. We examined the cross-sensory interplay by applying a quiet and motion-tolerant zero echo time functional magnetic resonance imaging (fMRI) technique to elucidate the evoked brain-wide responses to whisker pad stimulation in awake and anesthetized rats. Specifically, characterized the brain-wide responses in core and non-core regions to whisker pad stimulation by the varying stimulation-frequency, and determined whether isoflurane-medetomidine anesthesia, traditionally used in preclinical imaging, confounded investigations related to sensory integration. We demonstrated that unilateral whisker pad stimulation not only elicited robust activity along the whisker-mediated tactile system, but also in auditory, visual, high-order, and cerebellar regions, indicative of brain-wide cross-sensory and associative activity. By inspecting the response profiles to different stimulation frequencies and temporal signal characteristics, we observed that the non-core regions responded to stimulation in a very different way compared to the primary sensory system, likely reflecting different encoding modes between the primary sensory, cross-sensory, and integrative processing. Lastly, while the activity evoked in low-order sensory structures could be reliably detected under anesthesia, the activity in high-order processing and the complex differences between primary, cross-sensory, and associative systems were visible only in the awake state. We conclude that our study reveals novel aspects of the cross-sensory interplay of whisker-mediated tactile system, and importantly, that these would be difficult to observe in anesthetized rats.

neuroscience↗

bioRxiv2024

Raffinose induces autophagy to promote plant growth

Plant growth is governed by the integration of environmental cues and nutritional status. Under stress conditions, growth is usually attenuated in favor of stress response, creating a trade-off between growth and stress. Autophagy is a vital process in eukaryotes, maintaining cellular balance by degrading and recycling cellular components. It is triggered by various nutrient-deprivation conditions and both biotic and abiotic stresses in plants. Surprisingly, over-expressing autophagy-related genes across multiple plant species resulted in increased plant size, yield, and stress resistance, posing autophagy as a regulator of the stress-growth balance. Yet, the molecular mechanisms governing its induction remain partially understood. In the current work, we identified raffinose-a plant-derived sugar known for its role in stress responses-as a novel plant autophagy inducer. Raffinose treatment resulted in increased biomass and yield in an autophagy-dependent manner in several plant species. We also show that raffinose activates autophagy through the SnRK1 kinase complex, independent of TOR signaling, and that raffinose treatment results in increased expression of ATG5 and ATG7. We also point to possible downstream candidates operating autophagy-related biomass accumulation. Our findings offer new perspectives on the role of autophagy in maintaining a balance between plant growth and stress responses, underscoring the significance of raffinose in its regulation. SIGNIFICANCE STATEMENTThe intricate balance between plant growth and stress responses is crucial for agricultural productivity, particularly as climate change intensifies environmental stressors such as drought and extreme temperatures. Usually, there is a trade-off between growth and stress response. Autophagy--a cellular recycling process essential for maintaining cellular homeostasis--plays a pivotal role in this balance. Yet, the molecular mechanisms modulating it are partially understood. Raffinose treatment enhances biomass and yields in various plant species by inducing autophagy. By elucidating the molecular mechanisms of raffinose-mediated autophagy induction, our findings provide valuable insights into potential strategies for enhancing plant resilience against climate-induced stress.

plant biology↗

bioRxiv2023

Autophagy Restricts Tomato Fruit Ripening Via a General Role in Ethylene Repression

Autophagy, a cellular degradation pathway, and the phytohormone ethylene function in plant development, senescence, and stress responses. However, the manner of their interaction is mostly unknown. We reasoned that this may be revealed by studying autophagy in a climacteric fruit ripening context, for which ethylene is crucial. During ripening, fruits undergo softening, color change, toxic compound degradation, volatile production, and sugar assembly by fine-tuning synthesis and degradation of their cellular content. For autophagy activity assessment, we analyzed autophagy-related 8 (ATG8) lipidation and GFP-ATG8-labeled autophagosome flux in tomato fruit cells. Autophagy activity increased sharply from ripening initiation, climaxed at its middle stage, and declined towards its end, resembling ethylene production dynamics. Silencing the core-autophagy genes SlATG2, SlATG7, and SlATG4 separately in mature fruits resulted in early ethylene production and ripening onset, which was abrogated by 1-methylcyclopropene (1-MCP), an ethylene signaling inhibitor. Beyond ripening, Arabidopsis atg5 and atg7 mutant seedlings exhibited elevated ethylene production and sensitivity to 1-Aminocyclopropane 1-carboxylic acid (ACC), ethylenes precursor, which induces autophagy. This research demonstrates that autophagy limits tomato fruit ripening via a general role in ethylene restriction, opening the path for a mechanistic understanding of autophagy-ethylene crosstalk and harnessing autophagy for fruit shelf-life extension.

plant biology↗

bioRxiv2023

Self-eating while being eaten: Elucidating the relationship between aphid feeding and the plant autophagy machinery in Arabidopsis leaves

Autophagy, an intracellular process that facilitates the degradation of cytoplasmic materials, plays a dominant role in plant fitness and immunity. While autophagy was shown to be involved in plant response to fungi, bacteria, and viruses, its role in response to insect herbivory is as yet unknown. In this study, we demonstrate a role of autophagy in plant defense against herbivory using Arabidopsis thaliana and the green peach aphid, Myzus persicae. Following six hours of aphid infestation of wildtype plants, we observed high expression of the autophagy-related genes ATG8a and ATG8f, as well as NBR1 (Next to BRCA1 gene 1), a selective autophagy receptor. Moreover, the number of autophagosomes detected by the overexpression of GFP-fused ATG8f in Arabidopsis increased upon aphid infestation. Following this, atg5.1 and atg7.2 mutants were used to study the effect of autophagy on aphid reproduction and feeding behavior. While aphid reproduction on both mutants was lower than on wildtype, feeding behavior was only affected by atg7.2 mutants. Moreover, upon aphid feeding, the Phytoalexin-deficient 4 (PAD4) defense gene was upregulated in wildtype plants but not affected in the mutants. By contrast, the hydrogen peroxide content was much higher in the mutants relative to wildtype, which might have disturbed aphid reproduction and interfered with their feeding. Additionally, an analysis of the phloem sap metabolite profile revealed that atg7.2 mutant plants have lower levels of amino acids and sugars. These findings, together with the high hydrogen peroxide levels, suggest that aphids might exploit the plant autophagy mechanism for their survival.

plant biology↗

bioRxiv2021

The spliced leader RNA silencing (SLS) pathway in Trypanosoma brucei is induced by perturbations of endoplasmic reticulum, Golgi, or mitochondrial proteins factors and functional analysis of SLS inducing kinase, PK3

In the parasite Trypanosoma brucei, the causative agent of human African sleeping sickness, all mRNAs are trans-spliced to generate a common 5 exon derived from the spliced leader RNA (SL RNA). Perturbations of protein translocation across the endoplasmic reticulum (ER) induce the spliced leader RNA silencing (SLS) pathway. SLS activation is mediated by a serine-threonine kinase, PK3, which translocates from the cytosolic face of the ER to the nucleus, where it phosphorylates the TATA binding protein TRF4, leading to the shut-off of SL RNA transcription, followed by induction of programmed cell death. Here, we demonstrate that SLS is also induced by depletion of the essential ER resident chaperones BiP and calreticulin, ER oxidoreductin 1 (ERO1), and the Golgi-localized quiescin sulfhydryl oxidase (QSOX1). Most strikingly, silencing of Rhomboid-like 1(TIMRHOM1) involved in mitochondrial protein import, also induces SLS. The PK3 kinase, which integrates SLS signals, is modified by phosphorylation on multiple sites. To determine which of the phosphorylation events activate PK3, several individual mutations or their combination were generated. These mutations failed to completely eliminate the phosphorylation or translocation of the kinase to the nucleus. The structure of PK3 kinase and its ATP binding domain were therefore modeled. A conserved phenylalanine at position 771 was proposed to interact with ATP, and the PK3F771L mutation completely eliminated phosphorylation under SLS, suggesting that the activation involves most if not all the phosphorylation sites. The study suggests that the SLS occurs broadly in response to failures in protein sorting, folding, or modification across multiple compartments.

molecular biology↗

bioRxiv2021

Whole-brain studies of spontaneous behavior in head-fixed rats enabled by zero echo time MB-SWIFT fMRI

Understanding the link between the brain activity and behavior is a key challenge in modern neuroscience. Behavioral neuroscience, however, lacks tools to record whole-brain activity in complex behavioral settings. Here we demonstrate that a novel Multi-Band SWeep Imaging with Fourier Transformation (MB-SWIFT) functional magnetic resonance imaging (fMRI) approach enables whole-brain studies in spontaneously behaving head-fixed rats. First, we show anatomically relevant functional parcellation. Second, we show sensory, motor, exploration, and stress-related brain activity in relevant networks during corresponding spontaneous behavior. Third, we show odor-induced activation of olfactory system with high correlation between the fMRI and behavioral responses. We conclude that the applied methodology enables novel behavioral study designs in rodents focusing on tasks, cognition, emotions, physical exercise, and social interaction. Importantly, novel zero echo time and large bandwidth approaches, such as MB-SWIFT, can be applied for human behavioral studies, allowing more freedom as body movement is dramatically less restricting factor.

neuroscience↗

bioRxiv2021

Post-transcriptional regulation of Leishmania fitness gain

The protozoan parasite Leishmania donovani causes fatal human visceral leishmaniasis in absence of treatment. Genome instability has been recognized as a driver in Leishmania fitness gain in response to environmental change or chemotherapy. How genome instability generates beneficial phenotypes despite potential deleterious gene dosage effects is unknown. Here we address this important open question applying experimental evolution and integrative systems approaches on parasites adapting to in vitro culture. Phenotypic analyses of parasites from early and late stages of culture adaptation revealed an important fitness tradeoff, with selection for accelerated growth in promastigote culture (fitness gain) impairing infectivity (fitness costs). Comparative genomics, transcriptomics and proteomics analyses revealed a complex regulatory network driving parasite fitness, with genome instability causing highly reproducible, gene dosage-dependent changes in protein abundance linked to post-transcriptional regulation. These in turn were associated with a gene dosage-independent reduction in abundance of flagellar transcripts and a coordinated increase in abundance of coding and non-coding RNAs implicated in ribosomal biogenesis and protein translation. We correlated differential expression of small nucleolar RNAs (snoRNAs) with changes in rRNA modification, providing first evidence that Leishmania fitness gain in culture may be controlled by post-transcriptional and epitranscriptomic regulation. Our findings propose a novel model for Leishmania fitness gain in culture, where differential regulation of mRNA stability and the generation of fitness-adapted ribosomes may potentially filter deleterious from beneficial gene dosage effects and provide proteomic robustness to genetically heterogenous, adapting parasite populations. This model challenges the current, genome-centric approach to Leishmania epidemiology and identifies the Leishmania transcriptome and non-coding small RNome as potential novel sources for the discovery of biomarkers that may be associated with parasite phenotypic adaptation in clinical settings.

microbiology↗

bioRxiv2020

Lytic Reactivation of the Kaposis sarcoma-associated herpesvirus (KSHV) is Accompanied by Major Nucleolar Alterations

The nucleolus is a sub-nuclear compartment whose primary function is the biogenesis of ribosomal subunits. Certain viral infections affect the morphology and composition of the nucleolar compartment and influence ribosomal RNA (rRNA) transcription and maturation. However, no description of nucleolar morphology and function during infection with Kaposis sarcoma-associated herpesvirus (KSHV) is available to date. Using immunofluorescence microscopy, we documented extensive destruction of the nuclear and nucleolar architecture during lytic reactivation of KSHV. This was manifested by redistribution of key nucleolar proteins, including the rRNA transcription factor, UBF, the essential pre-rRNA processing factor Fibrillarin, and the nucleolar multifunctional phosphoproteins Nucleophosmin (NPM1) and Nucleolin. Distinct delocalization patterns were evident; certain nucleolar proteins remained together whereas others dissociated, implying that nucleolar proteins undergo nonrandom programmed dispersion. Of note, neither Fibrillarin nor UBF colocalized with promyelocytic leukemia (PML) nuclear bodies or with the viral protein LANA-1, and their redistribution was not dependent on viral DNA replication or late viral gene expression. No significant changes in pre-rRNA levels and no accumulation of pre-rRNA intermediates were found by RT-qPCR and Northern blot analysis, respectively. Furthermore, fluorescent in situ hybridization (FISH), combined with immunofluorescence, revealed an overlap between Fibrillarin and internal transcribed spacer 1 (ITS1), which represents the primary product of the pre-rRNA, suggesting that the processing of rRNA proceeds during lytic reactivation. Finally, small changes in the levels of pseudouridylation were documented across the rRNA. Taken together, our results suggest that rRNA transcription and processing persist during lytic reactivation of KSHV, yet they may become uncoupled. Whether the observed nucleolar alterations favor productive infection or signify cellular anti-viral responses remains to be determined. Author SummaryWe describe the extensive destruction of the nuclear and nucleolar architecture during lytic reactivation of KSHV. Distinct delocalization patterns are illustrated: certain nucleolar proteins remained associated with each other whereas others dissociated, implying that nucleolar proteins undergo nonrandom programmed dispersion. Of note, no significant changes in pre-rRNA levels and no accumulation of pre-rRNA intermediates were found, suggesting that pre-RNA transcription and processing continue and could be uncoupled during lytic reactivation. Small changes in the levels of pseudouridylation were documented across the rRNA. Previous studies showed that the different forms of KSHV infection are controlled through cellular and viral functions, which reprogram host epigenetic, transcriptomic, post-transcriptomic and proteomic landscapes. The ability of KSHV to affect the nucleolus and rRNA modifications constitutes a novel interaction network between viral and cellular components. The study of rRNA modifications is still in its infancy; however, the notion of altering cell fate by regulating rRNA modifications has recently begun to emerge, and its significance in viral infection is intriguing.

microbiology↗

bioRxiv2020

ATG8-interacting (ATI) 1 and 2 define a plant starvation-induced ER-phagy pathway and serve as MSBP1 (MAPR5) cargo-receptors

ER-phagy, the selective autophagy of endoplasmic reticulum (ER) components, is known to operate in eukaryotes from yeast and unicellular algae to animals and plants. Thus far, only ER-stress derived ER-phagy was reported and analyzed in plants. In this study we characterize an ER-phagy pathway in Arabidopsis thaliana that is triggered by dark-induced starvation and not by ER-stress. This pathway is defined by the previously reported ATG8-interacting proteins, ATI1 and ATI2 and is regulated by the TOR signaling pathway. We further identified ER-localized Membrane Steroid Binding Protein 1 (MSBP1) as an ATI1 and 2 interacting protein and an autophagy cargo, and show that ATI1 and 2 serve as its cargo receptors. Together, these findings expand our knowledge on plants responses during energy deprivation and highlight the role of this special type of ER-phagy in this process.

plant biology↗