The 3D Architecture of Interphase Microtubule Cytoskeleton and Functions of Microtubule Plus End Tracking Proteins in Fission Yeast

The microtubule (MT) cytoskeleton is important for establishing polar growth in the rod-shaped fission yeast (Schizosaccharomyces pombe). In these cells, MTs form an architectural scaffold of the cell by positioning organelles such as the nucleus and mitochondria. Interphase MTs are arranged in bundles along the cell’s long axis. The filaments start growing in the cell’s middle in a zone of anti-parallel overlap, from which the more dynamic plus ends of MTs extend towards both cell ends. After cell division the cell grows exclusively from the old end (away from the septum), where the growth machinery is still present from the mother cell. New end take off (NETO) occurs after about a third of the way through the cell cycle, when F-actin has moved into the new end. From this point onwards maintenance of polar growth is MT independent and occurs at both cell ends. Guidance of the microtubules to the cell ends is performed by plus end tracking proteins (+TIPs), such as Tea1 and Tip1 (Clip-170). Tea1 is a landmark protein localizing to the cell ends. Tip1 is an anti-catastrophe factor that prevents MT depolymerization before the filament has reached the cell end. The delivery of Tip1 to MT ends is motors dependent and another +TIP, Mal3, anchors it at the MT end. Mal3 (EB1) stabilizes MTs, possibly by fortify its seem. Here we describe a large-scale, electron tomography investigation of wild-type (WT) S. pombe cells, including the first 3D reconstruction of a complete eukaryotic cell volume. Sufficient resolution to show both how many MTs there are in a bundle and their detailed architecture was achieved. Most cytoplasmic MTs are open at one end and capped at the other, providing evidence about their polarity. Electron-dense bridges between the MTs themselves and between MTs and the nuclear envelope were frequently observed. Finally, we have investigated structure/function relationships between MTs and both mitochondria and vesicles. Using the same approach, we then analyzed the bundle architechture in tip1Δ and mal3Δ mutants. MTs were half the length of WT in mal3Δ and a quarter the length of WT in tip1Δ. Further, there were less than half as many MTs in a bundle in tip1Δ then in WT. In contrast, mal3Δ bundles no difference in the amount of filaments in a bundle. However, structural differences of the MT lattice were observed in both mutants. The interaction between MTs and the spindle pole body was altered in both strains. Our analysis shows that electron tomography of well-preserved cells is ideally suited for describing fine ultrastructural details that were not visible with previous techniques

SPACA9 is a lumenal protein of human ciliary singlet and doublet microtubules

The cilium-centrosome complex contains triplet, doublet, and singlet microtubules. The lumenal surfaces of each microtubule within this diverse array are decorated by microtubule inner proteins (MIPs). Here, we used single-particle cryo-electron microscopy methods to build atomic models of two types of human ciliary microtubule: the doublet microtubules of multiciliated respiratory cells and the distal singlet microtubules of monoflagellated human spermatozoa. We discover that SPACA9 is a polyspecific MIP capable of binding both microtubule types. SPACA9 forms intralumenal striations in the B tubule of respiratory doublet microtubules and noncontinuous spirals in sperm singlet microtubules. By acquiring new and reanalyzing previous cryo-electron tomography data, we show that SPACA9-like intralumenal striations are common features of different microtubule types in animal cilia. Our structures provide detailed references to help rationalize ciliopathy-causing mutations and position cryo-EM as a tool for the analysis of samples obtained directly from ciliopathy patients

A Novel Mouse Synaptonemal Complex Protein Is Essential for Loading of Central Element Proteins, Recombination, and Fertility

The synaptonemal complex (SC) is a proteinaceous, meiosis-specific structure that is highly conserved in evolution. During meiosis, the SC mediates synapsis of homologous chromosomes. It is essential for proper recombination and segregation of homologous chromosomes, and therefore for genome haploidization. Mutations in human SC genes can cause infertility. In order to gain a better understanding of the process of SC assembly in a model system that would be relevant for humans, we are investigating meiosis in mice. Here, we report on a newly identified component of the murine SC, which we named SYCE3. SYCE3 is strongly conserved among mammals and localizes to the central element (CE) of the SC. By generating a Syce3 knockout mouse, we found that SYCE3 is required for fertility in both sexes. Loss of SYCE3 blocks synapsis initiation and results in meiotic arrest. In the absence of SYCE3, initiation of meiotic recombination appears to be normal, but its progression is severely impaired resulting in complete absence of MLH1 foci, which are presumed markers of crossovers in wild-type meiocytes. In the process of SC assembly, SYCE3 is required downstream of transverse filament protein SYCP1, but upstream of the other previously described CE–specific proteins. We conclude that SYCE3 enables chromosome loading of the other CE–specific proteins, which in turn would promote synapsis between homologous chromosomes