Biology subjects

Nicastro, D.

Publications and source records attributed to Nicastro, D..

18 recordsLinked to original sources

bioRxiv2026

Unraveling Subcellular Ultrastructure with Cyclically Multiplexed Expansion Microscopy

Despite advances in fluorescence microscopy, spectral overlap and limited resolution hinder the dense mapping of the cellular ultrastructure. To overcome these challenges, we developed Cy-ExM, a high-plex imaging strategy that integrates optimized cryo-fixation for antigen preservation, expansion microscopy, and iterative immunofluorescence labeling. Using oblique plane microscopy, we perform three-dimensional super-resolution imaging of 20 biological targets encompassing the full cellular volume of individual mammalian cells.

cell biology↗

bioRxiv2025

The cryo-EM structure of mouse radial spoke 3 reveals a unique metabolic and regulatory hub in cilia

Cilia are complex, microtubule-based organelles that protrude from many eukaryotic cells and have important roles in sensing, signaling, and motility. Recent studies have revealed the atomic structures of many multi-component ciliary complexes, providing new insights into their mechanisms of action that are vital for cilias biological functions. However, little is known about the structure, proteome, and function of full-length radial spoke 3 (RS3), which is distinct from the structurally well-characterized RS1 and RS2. Radial spokes are conserved megadalton complexes that transmit mechanochemical signals from the central pair of microtubules to the dynein motors, thereby coordinating ciliary motility. Here, we combined cryo-electron microscopic single-particle reconstruction, cryo-electron tomography (cryo-ET), proteomic analysis, and computational modeling to determine the 3D structure and atomic model of RS3 from mouse respiratory cilia. Our structure reveals all protein components of RS3, including regulatory and metabolic enzymes, such as a protein kinase A subunit, adenylate kinases and malate dehydrogenases. We have confirmed the important role of adenylate kinase 7 in RS3 by cryo-ET analyses of respiratory cilia in AK7-deficient mice, which display primary ciliary dyskinesia. Our findings suggest that RS3 is an important regulatory hub and cluster of metabolic proteins that helps to maintain ATP at the levels required for sustained dynein motor activity and ciliary beating. This work advances our understanding of the structure and function of RS3 in ciliary motility and provides insights into the etiology of ciliopathies.

biophysics↗

bioRxiv2023

The ciliary MBO2 complex targets assembly of inner arm dynein b and reveals additional doublet microtubule asymmetries

Ciliary motility requires the spatiotemporal coordination of multiple dynein motors by regulatory complexes located within the 96 nm axoneme repeat. Many organisms can alter ciliary waveforms in response to internal or external stimuli, but little is known about the specific polypeptides and structural organization of complexes that regulate waveforms. In Chlamydomonas, several mutations convert the ciliary waveform from an asymmetric, ciliary-type stroke to a symmetric, flagellar-type stroke. Some of these mutations alter subunits located at the inner junction of the doublet microtubule and others alter interactions between the dynein arms and the radial spokes. These and other axonemal substructures are interconnected by a network of poorly characterized proteins. Here we re-analyze several motility mutants (mbo, fap57, pf12/pacrg) to identify new components in this network. The mbo (move backwards only) mutants are unable to swim forwards with an asymmetric waveform. Proteomics identified more than 19 polypeptides that are missing or reduced in mbo mutants, including one inner dynein arm, IDA b. Several MBO2-associated proteins are also altered in fap57 and pf12/parcg mutants, suggesting overlapping networks. Two subunits are highly conserved, coiled coil proteins found in other species with motile cilia and others contain potential signaling domains. Cryo-electron tomography and epitope tagging revealed that the MBO2 complex is found on specific doublet microtubules and forms a large, L-shaped structure that contacts the base of IDA b that interconnects multiple dynein regulatory complexes and varies in a doublet microtubule specific fashion.

cell biology↗

bioRxiv2023

Cryo-electron tomography of eel sperm flagella reveals a molecular "minimum system" for motile cilia

Cilia and flagella play a crucial role in the development and function of most eukaryotic organisms. The structural core of the flagellum, the axoneme, is conserved in most eukaryotes and careful regulation of dynein motors within the axoneme is required for proper ciliary beating. The sperm flagellum from the American eel (Anguilla rostrata) has been shown to lack many of the canonical axonemal proteins, including the radial spokes, the central pair complex, and possibly even the outer row of dynein arms, presenting a "minimal" flagellar system. Here, we present cryo-electron tomography analysis of the eel sperm flagellum. We identified two states for the eel sperm flagellum within our tomograms, narrow and wide, and found that the flagellum started narrow near the sperm head and widened distally. Subtomogram averages revealed that the eel sperm flagellum has retained remnants of the missing regulatory complexes, including a short radial spoke 3 complex, basal components of radial spokes 1 and 2, and an outer dynein arm docking complex. We also describe unique structural features of the A. rostrata sperm flagellum, such as a unique pattern of holes at the inner junction and an accessory complex located at the "outer" junction. Finally, we discuss the consequences of losing key regulatory factors for the eel sperm flagellum and hypothesize several evolutionary factors that may have led to their loss. Together, our results shed light onto the structure and function of the eel sperm axoneme and provide insight into the minimum requirements for proper ciliary beating.

cell biology↗

bioRxiv2022

Cryo-electron tomography of the apicomplexan invasion machinery in its native state reveals rigid body motion of the conoid and docked secretory machinery

The apical complex is a specialized collection of cytoskeletal and secretory machinery in apicomplexan parasites, which include the pathogens that cause malaria and toxoplasmosis. Its structure and mechanism of motion are poorly understood. We used cryo-FIB-milling and cryo-electron tomography to visualize the 3D-structure of the apical complex in its protruded and retracted states. Averages of conoid-fibers revealed their polarity and unusual nine-protofilament arrangement with associated proteins connecting and likely stabilizing the fibers. Neither the structure of the conoidfibers nor the architecture of the spiral-shaped conoid complex change during protrusion or retraction. Thus, the conoid moves as a rigid body, and is not spring-like and compressible, as previously suggested. Instead, the apical-polar-rings (APR), previously considered rigid, dilate during conoid protrusion. We identified actin-like filaments connecting the conoid and APR during protrusion, suggesting a role during conoid movements. Furthermore, our data capture the parasites in the act of secretion during conoid protrusion.

biophysics↗

bioRxiv2022

Triglyceride lipolysis driven by glucose restriction triggers liquid-crystalline phase transitions and proteome remodeling of lipid droplets

Lipid droplets (LDs) are reservoirs for triglycerides (TGs) and sterol-esters (SEs), but how these lipids are organized within LDs and influence its proteome remains unclear. Using in situ cryoelectron tomography, we show that glucose restriction triggers lipid phase transitions within LDs generating liquid-crystalline lattices inside them. Mechanistically this requires TG lipolysis, which decreases the LD TG:SE ratio, promoting SE transition to a liquid-crystalline phase. Molecular dynamics simulations reveal TG depletion promotes spontaneous TG and SE de-mixing in LDs, additionally altering the lipid packing of the phospholipid monolayer surface. Fluorescence imaging and proteomics further reveal that liquid-crystalline phases are associated with selective remodeling of the LD proteome. Some canonical LD proteins including Erg6 re-localize to the ER network, whereas others remain LD-associated. Model peptide LiveDrop also redistributes from LDs to the ER, suggesting liquid-crystalline-phases influence ER-LD inter organelle transport. Our data suggests glucose restriction drives TG mobilization, which alters the phase properties of LD lipids and selectively remodels the LD proteome.

cell biology↗

bioRxiv2022

Three-dimensional cilia structures from animals' closest unicellular relatives, the Choanoflagellates

In most eukaryotic organisms, cilia perform a variety of life-sustaining roles related to environmental sensing and motility. Cryo-electron microscopy has provided considerable insight into the morphology and function of ciliary structures, but studies have been limited to less than a dozen of the millions of known eukaryotic species. Ultrastructural information is particularly lacking for unicellular organisms in the opisthokont clade, leaving a sizeable gap in our understanding of cilia evolution between unicellular species and multicellular metazoans (animals). Choanoflagellates are important aquatic heterotrophs, uniquely positioned within the opisthokonts as the metazoans closest living unicellular relatives. We performed cryo-focused ion beam milling and cryo-electron tomography on cilia from the choanoflagellate species Salpingoeca rosetta. We show that the axonemal dyneins, radial spokes, and central pair complex in S. rosetta more closely resemble metazoan structures than those of unicellular organisms from other suprakingdoms. In addition, we describe unique features of S. rosetta cilia, including microtubule holes, microtubule inner proteins, and the ciliary vane: a fine, net-like extension that has been notoriously difficult to visualize using other methods. Furthermore, we report barb-like structures of unknown function on the extracellular surface of the ciliary membrane. Together, our findings provide new insights into choanoflagellate biology and cilia evolution between unicellular and multicellular opisthokonts.

cell biology↗

bioRxiv2021

Liquid-crystalline lipid phase transitions in lipid droplets selectively remodel the LD proteome

Lipid droplets (LDs) are reservoirs for triglycerides (TGs) and sterol-esters (SEs). How lipids are organized within LDs and influence the LD proteome remains unclear. Using in situ cryo-electron tomography, we show that glucose restriction triggers lipid phase transitions within LDs generating liquid-crystalline lattices inside them. Mechanistically, this requires TG lipolysis, which alters LD neutral lipid composition and promotes SE transition to a liquid-crystalline phase. Fluorescence imaging and proteomics further reveal that LD liquid-crystalline lattices selectively remodel the LD proteome. Some canonical LD proteins including Erg6 re-localize to the ER network, whereas others remain on LDs. Model peptide LiveDrop also redistributes from LDs to the ER, suggesting liquid-crystalline-phases influence ER-LD inter-organelle transport. Proteomics also indicates glucose restriction elevates peroxisome lipid oxidation, suggesting TG mobilization provides fatty acids for cellular energetics. This suggests glucose restriction drives TG mobilization, which alters the phase properties of LD lipids and selectively remodels the LD proteome.

cell biology↗

bioRxiv2021

3D structure and in situ arrangements of CatSper channel in the sperm flagellum

The sperm calcium channel CatSper plays a central role in successful fertilization as a primary Ca2+ gateway into the sperm flagellum. However, CatSpers complex subunit composition has impeded its reconstitution in vitro and structural elucidation. Here, we applied cryo-electron tomography to visualize the macromolecular organization of the native CatSper channel complex in intact mammalian sperm, as well as identified three additional CatSper-associated proteins. The repeating CatSper units form long zigzag-rows in four nanodomains along the flagella. In both mouse and human sperm, each CatSper repeat consists of a tetrameric pore complex. Murine CatSper contains an additional outwardly directed wing-structure connected to the tetrameric channel. The majority of the extracellular domains form a canopy above each pore-forming channel that interconnects to a zigzag-shaped roof. The intracellular domains link two neighboring channel complexes to a diagonal array. The loss of this intracellular link in Efcab9-/- sperm distorts the longitudinally aligned zigzag pattern and compromises flagellar movement. This work offers unique insights into the mechanisms underlying the assembly and transport of the CatSper complex to generate the nanodomains and provides a long-sought structural basis for understanding CatSper function in the regulation of sperm motility.

biophysics↗

bioRxiv2021

Structural organization of the C1b projection within the ciliary central apparatus

9+2 motile cilia contain 9 doublet microtubules and a central apparatus (CA) composed of two singlet microtubules with associated projections. The CA plays crucial roles in regulating ciliary motility. Defects in CA assembly or function usually result in motility-impaired or paralyzed cilia, which in humans causes disease. Despite their importance, the protein composition and functions of most CA projections remain largely unknown. Here, we combined genetic approaches and quantitative proteomics with cryo-electron tomography and subtomogram averaging to compare the CA of wild-type Chlamydomonas with those of two CA mutants. Our results show that two conserved proteins, FAP42 and FAP246, are localized to the L-shaped C1b projection of the CA. We also identified another novel CA candidate protein, FAP413, which interacts with both FAP42 and FAP246. FAP42 is a large protein that forms the peripheral beam of the C1b projection, and the FAP246-FAP413 subcomplex serves as the bracket between the beam (FAP42) and the C1b pillar that attaches the projection to the C1 microtubule. The FAP246-FAP413-FAP42 complex is essential for stable assembly of both the C1b and C1f projections, and loss of any of these proteins leads to ciliary motility defects. Our results provide insight into the subunit organization and 3D structure of the C1b projection, suggesting that the FAP246-FAP413-FAP42 subcomplex is part of a large interconnected CA-network that provides mechanical support and may play a role in mechano-signaling between the CA and radial spokes to regulate dynein activity and ciliary beating. Summary StatementThe present work provides insight into the subunit organization and 3D structure of the C1b projection of CA and the mechanism by which it regulates dynein activity and ciliary beating.

cell biology↗

bioRxiv2020

Proteomic analysis of microtubule inner proteins (MIPs) in Rib72 null Tetrahymena cells reveals functional MIPs

Motile cilia and flagella are built from stable populations of doublet microtubules that comprise their axonemes. Their unique stability is brought about, at least in part, by a network of Microtubule Inner Proteins (MIPs) found in the lumen of their doublet microtubules. Rib72A and Rib72B were identified as microtubule inner proteins (MIPs) in the motile cilia of Tetrahymena thermophila. Loss of these proteins leads to ciliary defects and loss of multiple MIPs. We performed mass spectrometry coupled with proteomic analysis and bioinformatics to identify the MIPs lost in RIB72A/B knockout (KO) Tetrahymena cells. From this analysis we identified a number of candidate MIPs and pursued one, Fap115, for functional characterization. We find that loss of Fap115 results in disrupted cell swimming and aberrant ciliary beating. Cryo-electron tomography reveals that Fap115 localizes to MIP6a in the A-tubule of the doublet microtubules. Overall, our results highlight the complex relationship between MIPs, ciliary structure, and ciliary function.

molecular biology↗

bioRxiv2019

Structural organization of the C1a-e-c supercomplex within the ciliary central apparatus

Nearly all motile cilia contain a central apparatus (CA) composed of two connected singlet-microtubules with attached projections that play crucial roles in regulating ciliary motility. Defects in CA assembly usually result in motility-impaired or paralyzed cilia, which in humans causes disease. Despite their importance, the protein composition and functions of the CA projections are largely unknown. Here, we integrated biochemical and genetic approaches with cryo-electron tomography to compare the CA of wild type Chlamydomonas with CA mutants. We identified a large (>2 MDa) complex, the C1a-e-c supercomplex, that requires the PF16 protein for assembly and contains the CA components FAP76, FAP81, FAP92, and FAP216. We localized these subunits within the supercomplex using nanogold-labeling and show that loss of any one of them results in impaired ciliary motility. These data provide insight into the subunit organization and three-dimensional (3D) structure of the CA, which is a prerequisite for understanding the molecular mechanisms by which the CA regulates ciliary beating.\n\nSummaryFu et al. use a wild-type vs. mutant comparison and cryo-electron tomography of Chlamydomonas flagella to identify central apparatus (CA) subunits and visualize their location in the native 3D CA structure. The study provides a better understanding of the CA and how it regulates ciliary motility.

cell biology↗

bioRxiv2019

FAP57/WDR65 targets assembly of a subset of inner arm dyneins and connects to regulatory hubs in cilia

Ciliary motility depends on both the precise spatial organization of multiple dynein motors within the 96 nm axonemal repeat, and highly coordinated interactions between different dyneins and regulatory complexes located at the base of the radial spokes. Mutations in genes encoding cytoplasmic assembly factors, intraflagellar transport factors, docking proteins, dynein subunits, and associated regulatory proteins can all lead to defects in dynein assembly and ciliary motility. Significant progress has been made in the identification of dynein subunits and extrinsic factors required for pre-assembly of dynein complexes in the cytoplasm, but less is known about the docking factors that specify the unique binding sites for the different dynein isoforms on the surface of the doublet microtubules. We have used insertional mutagenesis to identify a new locus, IDA8/BOP2, required for targeting the assembly of a subset of inner dynein arms to a specific location in the 96 nm repeat. IDA8 encodes FAP57/WDR65, a highly conserved WD repeat, coiled coil domain protein. Using high resolution proteomic and structural approaches, we find that FAP57 forms a discrete complex. Cryo-electron tomography coupled with epitope tagging and gold labeling reveal that FAP57 forms an extended structure that interconnects multiple inner dynein arms and regulatory complexes.

cell biology↗

bioRxiv2019

Scaffold subunits support associated subunit assembly in the Chlamydomonas ciliary nexin-dynein regulatory complex

The nexin-dynein regulatory complex (N-DRC) in motile cilia and flagella functions as a linker between neighboring doublet microtubules, acts to stabilize the axonemal core structure, and serves as a central hub for the regulation of ciliary motility. Although the N-DRC has been studied extensively using genetic, biochemical, and structural approaches, the precise arrangement of the eleven (or more) N-DRC subunits remains unknown. Here, using cryo-electron tomography, we have compared the structure of Chlamydomonas wild-type flagella to that of strains with specific DRC subunit deletions or rescued strains with tagged DRC subunits. Our results show that DRC7 is a central linker subunit that helps connect the N-DRC to the outer dynein arms. DRC11 is required for the assembly of DRC8, and DRC8/11 form a sub-complex in the proximal lobe of the linker domain that is required to form stable contacts to the neighboring B-tubule. Gold labeling of tagged subunits determines the precise locations of the previously ambiguous N-terminus of DRC4 which is now shown to contribute to the core scaffold of the N-DRC and C-terminus of DRC5. Our results reveal the overall architecture of N-DRC, with the three subunits, DRC1/2/4 forming a core complex that serves as the scaffold for the assembly of the \"functional subunits\" associate, namely DRC3/5-8/11. These findings shed light on N-DRC assembly and its role in regulating flagellar beating.\n\nSignificance StatementCilia and flagella are small hair-like appendages in eukaryotic cells that play essential roles in cell sensing, signaling, and motility. The highly conserved nexin-dynein regulatory complex (N-DRC) is one of the key regulators for ciliary motility. At least 11 proteins (DRC1-11) have been assigned to the N-DRC, but their precise arrangement within the large N-DRC structure is not yet known. Here, using cryo-electron tomography combined with genetic approaches, we have localized DRC7, the sub-complex DRC8/DRC11, the N-terminus of DRC4, and the C-terminus of DRC5. Our results provide insights into the N-DRC structure, its function in the regulation of dynein activity, and the mechanism by which n-drc mutations can lead to defects in ciliary motility that cause disease.

cell biology↗

bioRxiv2019

Complexity and ultrastructure of infectious extracellular vesicles from cells infected by nonenveloped virus

Enteroviruses support cell-to-cell viral transmission prior to their canonical lytic spread of virus. Poliovirus (PV), a prototype for human pathogenic positive-sense RNA enteroviruses, and picornaviruses in general, transport multiple virions en bloc via infectious extracellular vesicles secreted from host cells. Using biochemical and biophysical methods we identify multiple components in these secreted vesicles, including PV virions; positive and negative-sense viral RNA; essential viral replication proteins; ribosomal and regulatory cellular RNAs; and numerous host cell proteins, such as regulators of cellular metabolism and structural remodeling. Using cryo-electron tomography, we visualize the near-native three-dimensional architecture of secreted infectious extracellular vesicles containing both virions and a unique mat-like structure. Based on our biochemical data (western blot, RNA-Seq, and mass spectrometry), these mat-like structures are expected to be comprised of unencapsidated RNA and proteins. Our data show that, prior to cell lysis, non-enveloped viruses are secreted within infectious vesicles that also transport viral and host RNAs and proteins.\n\nImportanceThe family of picornaviridae is comprised of small positive-sense RNA viruses, many of which are significant human pathogens. Picornaviruses exploit secreted extracellular vesicles for cell-to-cell viral transmission without cell lysis, and poliovirus serves as a model system for picornaviruses that are not protected by a surrounding membrane (non-enveloped viruses). The structure and contents of these vesicles secreted by virus-infected cells are described here. In addition to mature virions, these vesicles carry negative-sense, template viral RNA and essential replication proteins, as well as cellular resources from the host. Their complex contents may comprise an enhanced virulence factor for propagation of infection, and understanding their structure and function is helping elucidate the mechanism by which extracellular vesicles contribute to the spread of non-enveloped virus infection.

cell biology↗

bioRxiv2018

Structure of the ciliary axoneme at nanometer resolution reconstructed by TYGRESS

The resolution of subtomogram averages calculated from cryo-electron tomograms (cryo-ET) of crowded cellular environments is often limited due to signal loss in, and misalignment of the subtomograms. In contrast, single-particle cryo-electron microcopy (SP-cryo-EM) routinely reaches near-atomic resolution of isolated complexes. We developed a novel hybrid-method called \"TomographY-Guided 3D REconstruction of Subcellular Structures\" (TYGRESS) that combines cryo-ET with SP-cryo-EM to achieve close-to-nanometer resolution of complexes inside crowded environments. Using TYGRESS, we determined the native 3D structures of the intact ciliary axoneme with up to 12 [A] resolution. These results reveal many structures and details that were not visible by cryo-ET. TYGRESS is generally applicable to cellular complexes that are amenable to subtomogram averaging, bringing us a step closer to (pseudo-)atomic models of cells.\n\nOne Sentence SummaryA hybrid cryo-electron microscopy method reveals subcellular structures at unprecedented resolution.

cell biology↗

bioRxiv2018

Tetrahymena RIB72A and RIB72B are Microtubule Inner Proteins in the ciliary doublet microtubules

Doublet and triplet microtubules are essential and highly stable core structures of centrioles, basal bodies, cilia and flagella. In contrast to dynamic cytoplasmic microtubules, their luminal surface is coated with regularly arranged Microtubule Inner Proteins (MIPs). However, the protein composition and biological function(s) of MIPs remain poorly understood. Using genetic, biochemical and imaging techniques we identified Tetrahymena RIB72A and RIB72B proteins as ciliary MIPs. Fluorescence imaging of tagged RIB72A and RIB72B showed that both proteins co-localize to Tetrahymena cilia and basal bodies, but assemble independently. Cryo-electron tomography of RIB72A and/or RIB72B knockout strains revealed major structural defects in the ciliary A-tubule involving MIP1, MIP4 and MIP6 structures. The defects of individual mutants were complementary in the double mutant. All mutants had reduced swimming speed and ciliary beat frequencies, and high-speed video imaging revealed abnormal highly curved cilia during power stroke. Our results show that RIB72A and RIB72B are crucial for the structural assembly of ciliary A-tubule MIPs and are important for proper ciliary motility.\n\nSUMMARYMicrotubule Inner Proteins (MIPs) bind to the luminal surface of highly stable microtubules. Combining cell biology and cryo-electron tomography, Stoddard et al. show that RIB72A and RIB72B are conserved MIPs in ciliary doublet microtubules and that they are important for proper ciliary motility.

cell biology↗

bioRxiv2017

Targeted dynein inhibition generates flagellar beating

Motile cilia and flagella are highly conserved organelles that are essential for the normal development and health of many eukaryotes including humans. To reveal the molecular mechanism of motility, we used cryo-electron tomography of active sea urchin sperm flagella to directly visualize the macromolecular complexes and their structural changes during flagellar beating. We resolved distinct conformations of dynein motors and regulators, and showed that many of them are distributed in bend-direction-dependent fashion in active flagella. Our results provide direct evidence for the conformational switching predicted by the \"switch-point-hypothesis\". However, they also reveal a fundamentally different mechanism of generating motility by inhibiting dyneins, rather than activating them, causing an asymmetric distribution of force and thus bending. Our high-resolution structural and biochemical analyses provide a new understanding of the distinct roles played by various dyneins and regulators in ciliary motility and suggest a molecular mechanism for robust beating in an all-or-none manner.\n\nHIGHLIGHTSO_LIDirect visualization of the switching mechanism for flagellar motility.\nC_LIO_LIFlagellar beating is generated by oscillating the side of dynein inhibition.\nC_LIO_LII1 dynein, I1-tether and N-DRC are key regulators that are involved in switching.\nC_LIO_LIStructural insights into distinct roles of dynein isoforms in flagellar motility.\nC_LI

cell biology↗