Biology subjects

Feric, M.

Publications and source records attributed to Feric, M..

7 recordsLinked to original sources

bioRxiv2026

Organization of core mitochondrial replication components into multiphasic condensates

Genomes are organized across several hierarchical levels. Intracellular phase transitions compartmentalize many genomic processes, including transcription and DNA repair. However, little is known regarding how phase transitions contribute to replication, in which long strands of double- and single-stranded DNA need to be coordinated. Here, we investigated the molecular interactions driving the condensation of core mitochondrial replication components into mt-nucleoids. Complex phase behavior emerged among purified mt-replication components: each nucleic acid colocalized with its cognate architectural protein within multiphasic condensates. Using single-molecule experiments, we found that formation of ssDNA increased the partitioning of its cognate protein mtSSB within the condensate, consistent with the preferential localization of mtSSB to replicating mt-nucleoids. To develop mechanistic insights, we built a minimalistic coarse-grained model of mt-replication components that showed how interactions between binary pairs dictate their assembly within condensates. The multiphasic organization of mt-nucleoids has implications for how replication can be spontaneously organized in cells. HighlightsO_LIComplex co-phase behavior of TFAM-mtDNA depends on their affinity and DNA length C_LIO_LIMt-replication architectural proteins segregate ssDNA and dsDNA within condensates C_LIO_LImtSSB selectively partitions into actively replicating nucleoids in vivo C_LIO_LIExposure of ssDNA promotes mtSSB partitioning into TFAM-mtDNA condensates C_LI

biophysics↗

bioRxiv2025

Transcription-dependent phase coexistence of mitochondrial nucleoids and RNA granules

The spatiotemporal organization of multiple components within biomolecular condensates helps coordinate gene expression. Mitochondria separate transcription and RNA processing into two distinct condensates: mt-nucleoids and mtRNA granules (MRGs), respectively. However, how mtRNA transcripts are transferred from mt-nucleoids to distant MRGs was unclear. With high-resolution imaging, we examined the steady-state organization of mt-condensates in human cells. We identified a wide distribution of distances between centroids of mt-condensates, from roughly a micron apart to within 100 nm of each other. Live imaging revealed that such organization was dynamic: mt-condensates frequently underwent cycles of mixing and demixing. Indeed, mtRNA transcripts and the mtRNA polymerase co-localized within mixed mt-condensates, while transcription inhibition led to complete dissolution of MRGs, supporting that nascently transcribed mtRNA is a key driver of mt-condensate organization. Together, our results show that active transcription sustains the phase coexistence between mt-nucleoids and MRGs, with implications for transcriptional condensates more broadly.

cell biology↗

bioRxiv2025

FLEXTAG: A Small and Self-renewable Protein Labeling System for Anti-fading Multi-color Super-resolution Imaging

Super-resolution fluorescence imaging enables visualization of subcellular structures and molecular interactions at the nanoscale, but its broader application has been hindered by long-standing limitations in current protein tagging systems, including rapid photobleaching, tag-induced artifacts, poor post-fixation labeling efficiency, and restricted multiplexing capability. Here, we present FLEXTAG (Fluorescent Labeling for Exchangeable, X-resilient Tagging in Advanced Generic Nanoscopy), a comprehensive protein labeling system comprising three orthogonal, ultrasmall (12-18 kDa), and self-renewable protein tags that collectively overcome these major limitations of existing tagging systems, enabling optimized multi-color super-resolution imaging. Through continuous exchange of organic fluorophores, FLEXTAG supports unprecedented durations of high-resolution imaging in both live and fixed cells with minimal photobleaching. It is compatible with major super-resolution modalities, such as SIM, STED, STORM, and PAINT, and is applicable to a wide range of subcellular targets. To further address fixation-induced labeling inefficiency and background fluorescence, we developed a novel protection-based fixation method and chemical blocking strategies that significantly preserve tag accessibility and enhance signal-to-noise ratio, improvements that are broadly applicable to other protein tagging systems. Altogether, FLEXTAG enables long-term tracking of dynamic behaviors and interactions of subcellular targets, as well as mapping of nanoscale protein organizations and cellular architecture, advancing both basic research and translational applications in cell biology.

bioengineering↗

bioRxiv2025

Membranes arrest the coarsening of mitochondrial condensates

Mitochondria contain double membranes that enclose their contents. Within their interior, the mitochondrial genome and its RNA products are condensed into [~]100 nm sized (ribo)nucleoprotein complexes. How these endogenous condensates maintain their roughly uniform size and spatial distributions within membranous mitochondria remains unclear. Here, we engineered an optogenetic tool (mt-optoIDR) that allowed for controlled formation of synthetic condensates upon light activation in live mitochondria. Using live cell super-resolution microscopy, we visualized the nucleation of small, yet elongated condensates (mt-opto-condensates), which recapitulated the morphologies of endogenous mitochondrial condensates. We decoupled the contribution of the double membranes from the environment within the matrix by overexpressing the dominant negative mutant of a membrane fusion protein (Drp1K38A). The resulting bulbous mitochondria had significantly more dynamic condensates that coarsened into a single, prominent droplet. These observations inform how mitochondrial membranes can limit the growth and dynamics of the condensates they enclose, without the need of additional regulatory mechanisms.

cell biology↗

bioRxiv2024

Global Release of Translational Repression Across Plasmodiums Host-to-Vector Transmission Event

Malaria parasites must be able to respond quickly to changes in their environment, including during their transmission between mammalian hosts and mosquito vectors. Therefore, before transmission, female gametocytes proactively produce and translationally repress mRNAs that encode essential proteins that the zygote requires to establish a new infection. This essential regulatory control requires the orthologues of DDX6 (DOZI), LSM14a (CITH), and ALBA proteins to form a translationally repressive complex in female gametocytes that associates with many of the affected mRNAs. However, while the release of translational repression of individual mRNAs has been documented, the details of the global release of translational repression have not. Moreover, the changes in spatial arrangement and composition of the DOZI/CITH/ALBA complex that contribute to translational control are also not known. Therefore, we have conducted the first quantitative, comparative transcriptomics and DIA-MS proteomics of Plasmodium parasites across the host-to-vector transmission event to document the global release of translational repression. Using female gametocytes and zygotes of P. yoelii, we found that nearly 200 transcripts are released for translation soon after fertilization, including those with essential functions for the zygote. However, we also observed that some transcripts remain repressed beyond this point. In addition, we have used TurboID-based proximity proteomics to interrogate the spatial and compositional changes in the DOZI/CITH/ALBA complex across this transmission event. Consistent with recent models of translational control, proteins that associate with either the 5 or 3 end of mRNAs are in close proximity to one another during translational repression in female gametocytes and then dissociate upon release of repression in zygotes. This observation is cross-validated for several protein colocalizations in female gametocytes via ultrastructure expansion microscopy and structured illumination microscopy. Moreover, DOZI exchanges its interaction from NOT1-G in female gametocytes to the canonical NOT1 in zygotes, providing a model for a trigger for the release of mRNAs from DOZI. Finally, unenriched phosphoproteomics revealed the modification of key translational control proteins in the zygote. Together, these data provide a model for the essential translational control mechanisms used by malaria parasites to promote their efficient transmission from their mammalian host to their mosquito vector.

microbiology↗

bioRxiv2022

Structure-function relationships in mitochondrial transcriptional condensates

Phase separation organizes many membraneless structures in cells. The functional consequences of concentrating cellular machinery into biomolecular condensates, however, are largely unclear. One fundamental cellular function that has been linked to condensate formation is transcription. Here, we have reconstituted mitochondrial transcription in condensates from purified components. We find that the core components of the mttranscriptional machinery form multi-phasic, viscoelastic condensates in vitro. Strikingly, the rates of condensate-mediated transcription are substantially lower than equivalent reactions in bulk solution. These condensate-mediated decreases in transcriptional rates are associated with the formation of dynamically arrested vesicular structures that are driven by the production and accumulation of RNA during transcription. Using coarse-grained, equilibrium simulations, we show that the generation of RNA alters the phase behavior and the organization of transcriptional components within condensates and that the in vitro mtcondensates are non-equilibrium structures. Together, our in vitro and in silico approaches shed light on how proteins and (ribo)nucleic acids biophysically self-assemble within mitochondria in vivo. Our results highlight the complex morphologies of transcribing, multicomponent condensates and they illustrate the interdependent structure-function relationships in condensates. Significance StatementMitochondria condense their genome into transcriptionally active mt-nucleoids. These structures fit the definition of biomolecular condensates that form via macromolecular phase separation. We take advantage of the ability to reconstitute mitochondrial transcriptional condensates in vitro from minimal components. We find that the production and accumulation of RNA alters the phase behavior of transcriptional condensates. The altered phase behavior is linked to the formation of arrested, non-equilibrium vesicular structures. Similar changes to phase behavior of proteins and (ribo)nucleic acids can be recapitulated in live mitochondria through knockdown of mt-nucleoid core components. Computer simulations help identify biophysical mechanisms that are needed to maintain the steady-state structures of transcriptional condensates.

biophysics↗

bioRxiv2019

Self-assembly of multi-component mitochondrial nucleoids via phase separation

Mitochondria contain an autonomous and spatially segregated genome. The organizational unit of their genome is the nucleoid, which consists of mitochondrial DNA (mtDNA) and associated architectural proteins. Here, we show that phase separation is the primary physical mechanism for assembly and size-control of the mitochondrial nucleoid. The major mtDNA-binding protein TFAM spontaneously phase separates in vitro via weak, multivalent interactions into viscoelastic droplets with slow internal dynamics. In combination, TFAM and mtDNA form multiphase, gel-like structures in vitro, which recapitulate the in vivo dynamic behavior of mt-nucleoids. Enlarged, phase-separated, yet transcriptionally active, nucleoids are present in mitochondria from patients with the premature aging disorder Hutchinson-Gilford Progeria Syndrome (HGPS) and are associated with mitochondrial dysfunction. These results point to phase separation as an evolutionarily conserved mechanism of genome organization. HighlightsO_LIMitochondrial genomes are organized by phase separation. C_LIO_LIThe main packaging protein TFAM and mtDNA combine to form viscoelastic, multiphase droplets in vitro. C_LIO_LIMitochondrial nucleoids exhibit phase behavior in vivo, including dynamic rearrangements and heterogenous organization. C_LIO_LICoalescence and enlargement of mt-nucleoids occur upon loss of mitochondrial homeostasis as well as in prematurely aged cells and are associated with mitochondrial dysfunction. C_LI

cell biology↗