scholarly journals Multivalent interactions facilitate motor-dependent protein accumulation at growing microtubule plus ends

2021 ◽  
Author(s):  
Renu Maan ◽  
Louis Reese ◽  
Vladimir A. Volkov ◽  
Matthew R. King ◽  
Eli van der Sluis ◽  
...  
Keyword(s):  
Liquid Phase ◽  
Electron Tomography ◽  
Protein Composition ◽  
Protein Accumulation ◽  
Disordered Protein ◽  
Cryo Electron Tomography ◽  
Dependent Protein ◽  
Multivalent Interactions ◽  
Disordered Networks ◽  
Mixed Protein

Growing microtubule ends provide platforms for the accumulation of plus-end tracking proteins that organize into comets of mixed protein composition. Using a reconstituted fission yeast system consisting of end-binding protein Mal3, kinesin Tea2 and cargo Tip1, we found that these proteins can be driven into liquid phase droplets both in solution and at microtubule ends under crowding conditions. In the absence of crowding agents, cryo-electron tomography revealed that motor-dependent comets consist of disordered networks where multivalent interactions appear to facilitate the non-stoichiometric accumulation of cargo Tip1. We dissected the contribution of two disordered protein regions in Mal3 and found that both are required for the ability to form droplets and Tip1 accumulation, while autonomous Mal3 comet formation only requires one of them. Using theoretical modeling, we explore possible mechanisms by which motor activity and multivalent interactions may lead to the observed enrichment of Tip1 at microtubule ends.

scholarly journals A cryo-ET study of microtubules in axons

2021 ◽  
Author(s):  
Helen E Foster ◽  
Camilla Ventura Santos ◽  
Andrew P Carter
Keyword(s):  
Drosophila Melanogaster ◽  
Dorsal Root ◽  
Cell Shape ◽  
Intracellular Transport ◽  
Electron Tomography ◽  
Structural Features ◽  
Disordered Protein ◽  
Cryo Electron Tomography ◽  
Mouse Dorsal Root Ganglion

The microtubule cytoskeleton in axons plays key roles in intracellular transport and in defining cell shape. Despite many years of study of microtubules, many questions regarding their native architecture remain unanswered. Here, we performed cryo-electron tomography of mouse dorsal root ganglion (DRG) and Drosophila melanogaster (Dm) neurons and examined their microtubule ultrastructure in situ. We found that the microtubule minus and plus ends in DRG axons are structurally similar and frequently contact nearby components. The microtubules in DRG axons maintained a 13 protofilament (pf) architecture, even close to lattice break sites. In contrast, microtubules in Dm neurons had 12 or 13 pfs and we detected sites of pf number transition. The microtubule lumen in DRG axons is filled with globular microtubule inner proteins (MIPs). Our data suggest these have a defined structure, which is surprising given they are thought to contain the disordered protein MAP6. In summary, we reveal novel morphological and structural features of microtubules in their native environment.

scholarly journals Structural organization of the C1b projection within the ciliary central apparatus

2021 ◽  
Author(s):  
Kai Cai ◽  
Yanhe Zhao ◽  
Lei Zhao ◽  
Nhan Phan ◽  
George Witman ◽  
...  
Keyword(s):  
Electron Tomography ◽  
3D Structure ◽  
Protein Composition ◽  
Ciliary Motility ◽  
Cryo Electron Tomography ◽  
Radial Spokes ◽  
Candidate Protein ◽  
Central Apparatus ◽  
Subtomogram Averaging ◽  
Subunit Organization

'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.

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

2019 ◽  
Author(s):  
Gang Fu ◽  
Lei Zhao ◽  
Erin Dymek ◽  
Yuqing Hou ◽  
Kangkang Song ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Electron Tomography ◽  
3D Structure ◽  
Three Dimensional ◽  
Protein Composition ◽  
Wild Type ◽  
Ciliary Motility ◽  
Cryo Electron Tomography ◽  
Central Apparatus ◽  
Subunit Organization

AbstractNearly 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.SummaryFu 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.

scholarly journals The flagellar motor of Vibrio alginolyticus undergoes major structural remodeling during rotational switching

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Brittany L Carroll ◽  
Tatsuro Nishikino ◽  
Wangbiao Guo ◽  
Shiwei Zhu ◽  
Seiji Kojima ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Electron Tomography ◽  
Protein Composition ◽  
Vibrio Alginolyticus ◽  
Flagellar Motor ◽  
Conformational Rearrangement ◽  
Structural Remodeling ◽  
Cryo Electron Tomography ◽  
Bacterial Flagellar Motor ◽  
Rotational Direction

The bacterial flagellar motor switches rotational direction between counterclockwise (CCW) and clockwise (CW) to direct the migration of the cell. The cytoplasmic ring (C-ring) of the motor, which is composed of FliG, FliM, and FliN, is known for controlling the rotational sense of the flagellum. However, the mechanism underlying rotational switching remains elusive. Here, we deployed cryo-electron tomography to visualize the C-ring in two rotational biased mutants in Vibrio alginolyticus. We determined the C-ring molecular architectures, providing novel insights into the mechanism of rotational switching. We report that the C-ring maintained 34-fold symmetry in both rotational senses, and the protein composition remained constant. The two structures show FliG conformational changes elicit a large conformational rearrangement of the rotor complex that coincides with rotational switching of the flagellum. FliM and FliN form a stable spiral-shaped base of the C-ring, likely stabilizing the C-ring during the conformational remodeling.

scholarly journals A cryo-ET survey of microtubules and intracellular compartments in mammalian axons

2021 ◽  
Vol 221 (2) ◽  
Author(s):  
Helen E. Foster ◽  
Camilla Ventura Santos ◽  
Andrew P. Carter
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Body ◽  
Visual Inspection ◽  
Electron Tomography ◽  
Disordered Protein ◽  
Sensory Axons ◽  
Cryo Electron Tomography ◽  
Intracellular Compartments ◽  
Membrane Bound ◽  
Subtomogram Averaging

The neuronal axon is packed with cytoskeletal filaments, membranes, and organelles, many of which move between the cell body and axon tip. Here, we used cryo-electron tomography to survey the internal components of mammalian sensory axons. We determined the polarity of the axonal microtubules (MTs) by combining subtomogram classification and visual inspection, finding MT plus and minus ends are structurally similar. Subtomogram averaging of globular densities in the MT lumen suggests they have a defined structure, which is surprising given they likely contain the disordered protein MAP6. We found the endoplasmic reticulum in axons is tethered to MTs through multiple short linkers. We surveyed membrane-bound cargos and describe unexpected internal features such as granules and broken membranes. In addition, we detected proteinaceous compartments, including numerous virus-like capsid particles. Our observations outline novel features of axonal cargos and MTs, providing a platform for identification of their constituents.

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

2019 ◽  
Vol 218 (12) ◽  
pp. 4236-4251 ◽  
Author(s):  
Gang Fu ◽  
Lei Zhao ◽  
Erin Dymek ◽  
Yuqing Hou ◽  
Kangkang Song ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Electron Tomography ◽  
3D Structure ◽  
Protein Composition ◽  
Ciliary Beating ◽  
Ciliary Motility ◽  
Cryo Electron Tomography ◽  
Central Apparatus ◽  
Subunit Organization ◽  
Insight Into

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 MD) 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 3D structure of the CA, which is a prerequisite for understanding the molecular mechanisms by which the CA regulates ciliary beating.

scholarly journals Ciliary Proteins: Filling the Gaps. Recent Advances in Deciphering the Protein Composition of Motile Ciliary Complexes

Cells ◽  
2019 ◽  
Vol 8 (7) ◽  
pp. 730 ◽  
Author(s):  
Anna Osinka ◽  
Martyna Poprzeczko ◽  
Magdalena M. Zielinska ◽  
Hanna Fabczak ◽  
Ewa Joachimiak ◽  
...  
Keyword(s):  
Primary Cilia ◽  
Higher Plants ◽  
Electron Tomography ◽  
Protein Complexes ◽  
Protein Composition ◽  
Model Organisms ◽  
Cryo Electron Tomography ◽  
Motile Cilia ◽  
Biochemical Analyses ◽  
Eukaryotic Organisms

Cilia are highly evolutionarily conserved, microtubule-based cell protrusions present in eukaryotic organisms from protists to humans, with the exception of fungi and higher plants. Cilia can be broadly divided into non-motile sensory cilia, called primary cilia, and motile cilia, which are locomotory organelles. The skeleton (axoneme) of primary cilia is formed by nine outer doublet microtubules distributed on the cilium circumference. In contrast, the skeleton of motile cilia is more complex: in addition to outer doublets, it is composed of two central microtubules and several diverse multi-protein complexes that are distributed periodically along both types of microtubules. For many years, researchers have endeavored to fully characterize the protein composition of ciliary macro-complexes and the molecular basis of signal transduction between these complexes. Genetic and biochemical analyses have suggested that several hundreds of proteins could be involved in the assembly and function of motile cilia. Within the last several years, the combined efforts of researchers using cryo-electron tomography, genetic and biochemical approaches, and diverse model organisms have significantly advanced our knowledge of the ciliary structure and protein composition. Here, we summarize the recent progress in the identification of the subunits of ciliary complexes, their precise intraciliary localization determined by cryo-electron tomography data, and the role of newly identified proteins in cilia.

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