Biology subjects

Bleck, C. K. E.

Publications and source records attributed to Bleck, C. K. E..

15 recordsLinked to original sources

bioRxiv2026

Organelle Volume Facilitates Spindle Positioning During C. elegans Female Meiosis

Cortical positioning of oocyte meiotic spindles is required for reducing chromosome number by segregating chromosomes into polar bodies. In C. elegans, kinesin-1 transports membrane-bound organelles inward into a central ball that is hypothesized to push the meiotic spindle outward. Using focused ion beam scanning electron microscopy, we found that the percent volume occupied by membrane-bound organelles was 28% in a mature oocyte that has completed endocytosis of the yolk precursor, vitellogenin, compared to 17% in a less mature oocyte that has not yet endocytosed vitellogenin. We therefore hypothesized that blocking endocytosis of vitellogenin would reduce the size of the central ball of organelles packed inward by kinesin-1 and limit outward movement of the spindle. Depletion of vitellogenin or proteins required for endocytosis resulted in a smaller central volume of kinesin-coated organelles and defective spindle positioning. A computer simulation of a simple exclusion model faithfully recapitulated wildtype and mutant spindle position phenotypes. These results support a volume exclusion model for positioning acentriolar meiotic spindles.

cell biology↗

bioRxiv2025

Alternating Angle Milling Suppresses Streaking Artifacts in FIB-SEM Imaging

Focused ion beam scanning electron microscopy (FIB-SEM) (1-3) has been used in life sciences to produce large volumetric datasets with high resolution information on ultrastructure of biological organisms. 3D image acquisition is accomplished by serial removal of thin layers of material using focused ion beam (FIB) milling followed by scanning electron microscopy (SEM) imaging. One of the challenges in the standard FIB-SEM imaging protocol is that FIB milling results in characteristic artifacts, known as "streaks" or "curtains". These streaks are caused by non-uniform material removal forming long straight trenches parallel to the FIB milling direction. These artifacts get worse along the milling direction and ultimately limit size of the SEM field of view. Various methods have been proposed to mitigate the streaks in acquired images. While these techniques often provide noticeable visual improvement, the underlying problem remains. The structural information in the "streaked" areas is lost due to non-uniform material removal during milling and cannot be fully recovered. We propose a simple modification allowing for a significant reduction of milling non-uniformities of streaks. We demonstrate the effectiveness of this approach on various samples.

cell biology↗

bioRxiv2025

Cdc42 regulates apical membrane fusion via the Rab11a-VAMP2 pathway in salivary gland acinar cells

Epithelial acinar cells of the salivary gland establish apical-basal polarity and secretory competence during development, processes that require tightly coordinated membrane trafficking. The Rho-family GTPase Cdc42 is a key regulator of epithelial polarity, but it has rarely been linked to the vesicle-fusion machinery in vivo. Using mice with acinar cell-specific deletion of Cdc42, we found that Cdc42 loss caused the accumulation of small vesicles near the apical surface, where the water channel AQP5 was retained in clusters and lumen formation was defective. Cdc42-deficient glands showed a selective reduction of two vesicle-associated SNAREs, VAMP2 and VAMP4, whereas VAMP8, the t-SNAREs, and an endocytic marker were unchanged. The corresponding mRNAs were not decreased, indicating that Cdc42 maintains these v-SNAREs through post-transcriptional mechanisms. Cdc42-deficient acinar cells also accumulated condensing vacuoles, a form of immature secretory granule, indicating impaired granule maturation. We propose that Cdc42 coordinates two distinct membrane-remodeling processes by maintaining these v-SNAREs: VAMP2 supports the fusion of AQP5-containing vesicles with the apical membrane, whereas VAMP4 supports secretory granule maturation. These findings identify the vesicle-fusion machinery as a target of Cdc42-dependent polarity control, with potential implications for secretory gland disorders.

cell biology↗

bioRxiv2024

Data Release: High-Resolution Imaging and Segmentation of P7 Mouse Tissue Microarchitecture Using FIB-SEM and Machine Learning

This report presents a comprehensive data release exploring the tissue microarchitecture of P7 aged mice using Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) combined with machine learning-based segmentations of nuclei. The study includes high-resolution 3D volumes and nucleus segmentations for seven vital tissues--pancreas, liver, kidney, heart, thymus, hippocampus, and skin--from a single mouse. The detailed datasets are openly accessible onOpenOrganelle.org, providing a valuable resource for the scientific community to support further research and collaboration.

cell biology↗

bioRxiv2022

Mitochondrial Network Configuration Influences Sarcomere and Myosin Filament Structure in Striated Muscles

Sustained muscle contraction occurs through interactions between actin and myosin filaments within sarcomeres and requires a constant supply of adenosine triphosphate (ATP) from nearby mitochondria. However, it remains unclear how different physical configurations between sarcomeres and mitochondria alter the energetic support for contractile function. Here, we show that sarcomere cross-sectional area (CSA) varies along its length in a cell type-dependent manner where the reduction in Z-disk CSA relative to the sarcomere center is closely coordinated with mitochondrial network configuration in flies, mice, and humans. Further, we find myosin filaments near the sarcomere periphery are curved relative to interior filaments with greater curvature for filaments near mitochondria compared to sarcoplasmic reticulum. Finally, we demonstrate smaller myosin filament lattice spacing at filament ends than filament centers in a cell type-dependent manner. These data suggest both sarcomere structure and myofilament interactions are influenced by the location and orientation of mitochondria within muscle cells.

cell biology↗

bioRxiv2021

OMA1 mediates local and global stress responses against protein misfolding in CHCHD10 mitochondrial myopathy

Mitochondrial stress triggers a response in the cells mitochondria and nucleus, but how these stress responses are coordinated in vivo is poorly understood. Here, we characterize a family with myopathy caused by a dominant p.G58R mutation in the mitochondrial protein CHCHD10. To understand the disease etiology, we developed a novel knock-in mouse model and found that mutant CHCHD10 aggregates in affected tissues, applying a toxic protein stress to the inner mitochondrial membrane. Unexpectedly, survival of CHCHD10 knock-in mice depended on a protective stress response mediated by OMA1. The OMA1 stress response acted both locally within mitochondria, inhibiting mitochondrial fusion, and signaled outside the mitochondria, activating the integrated stress response. We additionally identified an isoform switch in the terminal complex of the electron transport chain as a novel component of this response. Our results demonstrate that OMA1 is essential for neonatal survival conditionally in the setting of inner mitochondrial membrane stress, coordinating local and global stress responses to reshape the mitochondrial network and proteome. Graphical Abtract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/473493v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1ab5990org.highwire.dtl.DTLVardef@85080dorg.highwire.dtl.DTLVardef@1d3a036org.highwire.dtl.DTLVardef@bb4679_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

An Evolutionarily Conserved Regulatory Pathway of Muscle Mitochondrial Network Organization

Mitochondrial networks provide coordinated energy distribution throughout muscle cells. However, pathways specifying mitochondrial network-type separately from contractile fiber-type remain unclear. Here, we show that natural energetic demands placed on Drosophila melanogaster muscles yield native cell-types among which contractile and mitochondrial network-types are regulated independently. Proteomic analyses of indirect flight, jump, and leg muscles together with muscles misexpressing known fiber-type specification factor salm identified transcription factors H15 and cut as potential mitochondrial network regulators. We demonstrate H15 operates downstream of salm regulating flight muscle contractile and mitochondrial network-type. Conversely, H15 regulates mitochondrial network configuration but not contractile type in jump and leg muscles. Further, we find that cut regulates salm expression in flight muscles and mitochondrial network configuration in leg muscles. These data indicate cell type-specific regulation of muscle mitochondrial network organization separately from contractile type, mitochondrial content, and mitochondrial size through an evolutionarily conserved pathway involving cut, salm, and H15.

cell biology↗

bioRxiv2021

Regulation of the Evolutionarily Conserved Muscle Myofibrillar Matrix by Cell Type Dependent and Independent Mechanisms

Skeletal muscles play a central role in human movement through forces transmitted by contraction of the sarcomere. We recently showed that mammalian sarcomeres are connected through frequent branches forming a singular, mesh-like myofibrillar matrix. However, the extent to which myofibrillar connectivity is evolutionarily conserved as well as mechanisms which regulate the specific architecture of sarcomere branching remain unclear. Here, we demonstrate the presence of a myofibrillar matrix in the tubular, but not indirect flight (IF) muscles within Drosophila melanogaster. Moreover, we find that loss of transcription factor H15 increases sarcomere branching frequency in the tubular jump muscles, and we show that sarcomere branching can be turned on in IF muscles by salm-mediated conversion to tubular muscles. Finally, we demonstrate that neurochondrin knockdown results in myofibrillar connectivity in IF muscles without conversion to tubular muscles. These data indicate an evolutionarily conserved myofibrillar matrix regulated by both cell-type dependent and independent mechanisms.

cell biology↗

bioRxiv2021

Orchestrated Restructuring Events During Secretory Granule Maturation Mediate Intragranular Cargo Segregation

Regulated secretion is an essential process where proteins are packaged into membranous secretory vesicles. However, the details of cargo packaging and secretory granule maturation are largely unknown. Here, we demonstrate that multiple distinct proteins undergo intragranular restructuring during secretory granule maturation in vivo, resulting in spatial segregation of distinct protein components within the same granule. Furthermore, through a combination of genetics and multimodality imaging, we demonstrate the molecular identity of each distinct intragranular structure. We further identify temporally-regulated genes that are essential for the restructuring events, including those controlling pH (Vha16-1), Cl- ions (Clic and ClC-c) and Ca2+ ions (fwe). Finally, we show that altered cargo glycosylation influences dimensions of these structures, thereby affecting secretory granule morphology. This study elucidates key steps and factors involved in intragranular, rather than intergranular segregation of cargo through regulated restructuring events during secretory granule maturation. Understanding how multiple distinct proteins are efficiently packaged into and secreted from the same secretory granule may provide insight into diseases resulting from defects in secretion.

cell biology↗

bioRxiv2021

Reorganization of the Mitochondria-Organelle Interactome during Postnatal Development in Skeletal Muscle

Cellular development requires the integrated assembly of intracellular structures into functionally specialized regions supporting overall cellular performance. However, it remains unclear how coordination of organelle interactions contributes to development of functional specificity across cell types. Here, we utilize a subcellular connectomics approach to define the cell-scale reorganization of the mitochondria-organelle interactome across postnatal development in skeletal muscle. We show that while mitochondrial networks are disorganized and loosely associated with the contractile apparatus at birth, contact sites among mitochondria, lipid droplets, and the sarcoplasmic reticulum are highly abundant in neonatal muscles. The maturation process is characterized by a transition to highly organized mitochondrial networks wrapped tightly around the muscle sarcomere but also to less frequent interactions with both lipid droplets and the sarcoplasmic reticulum. These data demonstrate a developmental redesign reflecting a functional shift from muscle cell assembly supported by inter-organelle communication toward a muscle fiber highly specialized for contractile function.

physiology↗

bioRxiv2021

Metabolic design in a model of extreme mammalian metabolism, the North American least shrew (Cryptotis parva)

Mitochondrial adaptations are fundamental to differentiated function and energetic homeostasis in mammalian cells. But the mechanisms that underlie these relationships remain poorly understood. Here, we investigated organ-specific mitochondrial morphology, connectivity and protein composition in a model of extreme mammalian metabolism, the Least shrew (Cryptotis parva). This was achieved through a combination of high-resolution 3D focused-ion-beam EM imaging and tandem-mass-tag MS proteomics. We demonstrate that liver and kidney mitochondrial content are equivalent to the heart permitting assessment of mitochondrial adaptations in different organs with similar metabolic demand. Muscle mitochondrial networks (cardiac and skeletal) are extensive, with a high incidence of nanotunnels - which collectively support the metabolism of large muscle cells. Mitochondrial networks were not detected in the liver and kidney as individual mitochondria are localized with sites of ATP consumption. This configuration is not observed in striated muscle, likely due to a homogenous ATPase distribution and the structural requirements of contraction. These results demonstrate distinct, fundamental mitochondrial structural adaptations for similar metabolic demand that are dependent on the topology of energy utilization process in a mammalian model of extreme metabolism.

zoology↗

bioRxiv2020

Kinesin-1 motility traced by an activity-based precipitating dye

Kinesin-1 is a processive motor protein that uses ATP-derived energy to transport a variety of intracellular cargoes toward the cell periphery. As tracks for cargo delivery, kinesin-1 uses a subset of microtubules within the dense microtubule network. It is still debated what defines the specific binding of kinesin-1 to a subset of microtubules. Therefore, the ability to visualize and monitor kinesin transport in live cells is critical to study the myriad of functions associated with cargo trafficking. Herein we report the discovery of a fluorogenic small molecule substrate for kinesin-1 that yields a precipitating dye. The activity of kinesin-1 thus leaves a fluorescent trail along its walking path and can be visualized without loss of signal due to diffusion. Kinesin-1 specific transport of cargo from the Golgi appears as trails of fluorescence over time.

biochemistry↗

bioRxiv2020

LRRK2 mediates tubulation and vesicle sorting from membrane damaged lysosomes

Mutations in the leucine rich repeat kinase 2 (LRRK2) gene are a cause of familial and sporadic Parkinsons disease (PD). Nonetheless, the biological functions of LRRK2 remain incompletely understood. Here, we observed that LRRK2 is recruited to lysosomes that have a ruptured membrane. Using unbiased proteomics, we observed that LRRK2 is able to recruit the motor adaptor protein JIP4 to permeabilized lysosomes in a kinase-dependent manner through the phosphorylation of RAB35 and RAB10. Super-resolution live cell imaging microscopy and FIB-SEM revealed that once at the lysosomal membrane, JIP4 promotes the formation of LAMP1-negative lysosomal tubules that release membranous content from ruptured lysosomes. Released vesicular structures are able to interact with other lysosomes. Thus, we described a new process that uses lysosomal tubulation to release vesicular structures from permeabilized lysosomes. LRRK2 orchestrates this process that we name LYTL (LYsosomal Tubulation/sorting driven by LRRK2) that, given the central role of the lysosome in PD, is likely to be disease relevant.

cell biology↗

bioRxiv2019

Mutations in Auxilin cause parkinsonism via impaired clathrin-mediated trafficking at the Golgi apparatus and synapse

Parkinsons disease (PD) is a common neurodegenerative motor disorder characterized in part by neuropathological lesions in the nigrostriatal pathway. Loss of function mutations in Auxilin, the major neuronal clathrin uncoating protein, cause an aggressive form of juvenile onset PD. How mutations in Auxilin cause PD, is currently not understood. Here, we generated a novel mouse model carrying an endogenous pathogenic Auxilin mutation that phenocopies neurological features observed in patients, including motor impairments and seizures. Unbiased mapping of the Auxilin interactome identified synaptic and Golgi-resident clathrin adaptor proteins as novel interactors. Impaired clathrin-mediated trafficking in mutant Auxilin mice, both at the Golgi and the synapse, results in neuropathological lesions in the nigrostriatal pathway. Collectively, these results provide molecular mechanisms of PD pathogenesis in Auxilin mutation carriers, reinforcing a key role for clathrin-mediated trafficking in PD, and expand our understanding of the cellular function of Auxilin.

neuroscience↗

bioRxiv2016

Proteotype profiling unmasks a viral signaling network essential for poxvirus assembly and transcriptional competence

To orchestrate context-dependent signaling programs poxviruses encode two dual-specificity enzymes, the F10 kinase and the H1 phosphatase. These signaling mediators are essential for poxvirus production, yet their substrate profiles and systems level functions remain enigmatic. Using a phosphoproteomic screen of cells infected with wildtype, F10, and H1 mutant viruses we systematically defined the viral signaling network controlled by these enzymes. Quantitative cross-comparison revealed 33 F10 and/or H1 phosphosites within 17 viral proteins. Using this proteotype dataset to inform genotype-phenotype relationships we found that H1-deficient virions harbor a hidden hyper-cleavage phenotype driven by reversible phosphorylation of the virus protease I7 (S134). Quantitative phospho-proteotyping further revealed that the phosphorylation-dependent activity of the viral early transcription factor, A7 (Y367), underlies the transcription-deficient phenotype of H1 mutant virions. Together these results highlight the utility of combining quantitative proteotype screens with mutant viruses to uncover novel proteotype-phenotype-genotype relationships that are masked by classical genetic studies.

microbiology↗