Characterisation of the manchette architecture and its role as transport scaffold using cryo-electron tomography

Abstract

The development of correctly shaped sperm cells is crucial for male reproductive health and fertility. The manchette is a transient microtubule-based structure that assembles during spermiogenesis and contributes to sperm head shaping. Defects in the manchette can cause sperm deformations and subsequent infertility. Previous studies have suggested that the manchette acts as a cellular transport platform, distributing proteins and vesicles during spermiogenesis in a process known as intra-manchette transport. The manchette and intra-manchette transport are still poorly understood, as high-resolution imaging is missing. Here, we used cryo-electron tomography and proteomics to visualize the manchette and identify some of its transport components. We characterize the overall architecture of the manchette and show that its perinuclear ring thickens as the structure constricts. We observed for the first time dynein directly interacting with the manchette. We further find F-actin as single filaments and filament clusters intercalating with the manchette microtubules. Our results provide new insights into the manchette's architecture and potential role as a transport scaffold, highlighting its significance for the shaping of sperm cells during spermiogenesis.

Graphical abstract

Figure 1.. Vesicles colocalize with the manchette in intact spermatids.

(A) Workflow showing preparation of cryo-FIB–milled lamellae from mouse spermatids. (B) A tomographic slice showing the manchette as a 200-nm-wide array of MTs (green arrowheads) with its perinuclear ring close to the nucleus. Vesicles are seen close to MTs (blue arrowheads). (C) Tomographic slice showing mitochondria and vesicles colocalizing with MTs. (D) A tomographic slice revealing several vesicles and a filament (yellow arrowhead) between the manchette and the nucleus. Scale bars, 250 nm.

Figure 2.. Increasing thickness of the perinuclear ring correlates to manchette elongation and constriction.

(A, B, C) Projection images of rat manchettes with different dimensions on a cryo-EM grid and annotations below (perinuclear ring [PNR], dark grey; microtubule [MT] sheath, green). Scale bar, 2 μm. The box on the bottom left shows a zoom into the perinuclear ring with white triangles marking its thickness. Scale bar, 250 nm. (D, E) Tomographic slices showing the perinuclear ring of different thicknesses. Scale bars, 500 nm. (F) Quantification of perinuclear ring thickness across 90 different manchettes. (G) Plot of the perinuclear ring thickness relative to the manchette diameter (grey circles) and manchette length (green triangles). Lines show respective linear regressions.

Figure 3.. A membrane is laminated to the perinuclear ring.

(A) A tomographic slice resolving a membrane that envelopes the dense perinuclear ring (PNR) and vesicles that are associated with the microtubules. Scale bar, 250 nm. (B) Segmentation of the tomogram in (A). Membrane (brown), perinuclear ring (dark grey), and the MT sheath (green). (C) Distance measurements between the inner membrane leaflet and the top border of the perinuclear ring. (D) Tomographic slice revealing regular densities repeating every 8 nm under the membrane (white arrows). Scale bar, 25 nm. Annotation colour scheme: PNR, grey; membrane, brown; regular densities, teal.

Figure S1.. Observations of perinuclear ring thickness and integrity.

(A, B) Tomographic slices of manchettes with ripped membranes and broken perinuclear ring (white arrowheads) were excluded from measurements. Scale bars, 500 nm.

Figure S2.. Regular repeats on top of the perinuclear ring.

(A, B) Tomographic slices of the perinuclear ring revealing stripe-like arrays (between white triangles) close to the membrane. Scale bar, 100 nm.

Figure 4.. Vesicles are associated with the manchette.

(A) A low-magnification projection image of the manchette periphery revealing many vesicles colocalizing with MTs. Scale bar, 1 μm. (B, C) Tomographic slices showing many vesicles close to the MTs (blue triangles). Scale bars, 100 nm. (D, E, F) Tomographic slices (top) and annotation (bottom) showing vesicles (grey) connected to a microtubule (green) through a slender density (orange). Scale bars, 50 nm.

Figure 5.. Dynein on manchette MTs.

(A, B, C) Tomographic slices (top) and annotations (bottom) revealing densities associated with the MTs (green). Scale bars, 50 nm. (D) 3D reconstruction of the densities found in (A, B, C) with cytoplasmic dynein I motor domain (PDB: 5NVU) fitted into the map (right). (E) A slice through a reconstruction of the cytosolic dynein–dynactin complex (EMD-7000) with dynein motor domains coloured pink, and the dynein tails and dynactin complexes are coloured dark grey. (F) Relative abundance of tubulin, actin, and components of the transport machinery in isolated manchettes. Tubulins (green) and actins (red) show the highest abundance. Components of the microtubule motor proteins kinesins (orange) and dynein (pink) are similar in abundance. Components of the actin motor proteins myosins (yellow) are less abundant than dyneins and kinesins. IFT subunits (blue) show the lowest abundance.

Figure S3.. Fourier shell correlations (FSC) of subtomogram averages.

(A) FSC curve of subtomogram average of dynein motor domains. (B) FSC curve of subtomogram average of F-actin. Resolutions were estimated based on a cut-off at 0.5.

Figure S4.. MT protofilament skew indicates its directionality.

Green arrows show the direction of view. A clockwise protofilament skew is a view from the minus-end to plus-end direction. Scale bar, 50 nm.

Figure 6.. Actin filaments are intercalated in the manchette.

(A) Orthogonal slices from a tomogram showing non-MT filaments (yellow arrowheads and yellow circles). Scale bar, 50 nm. (B) 3D reconstruction and fitting of an F-actin structure (PDB: 7BT7). (C) Quantification of actin-to-actin filament distances. (D) Segmentation of the tomogram in (A) with actin filaments (yellow) running parallel and in between MTs (green). (E) Tomographic slice of a single actin filament (yellow arrowheads) bound by a large density (violet arrowheads). Scale bar, 50 nm. (F, G, H) Tomographic slices showing actin single filaments (yellow arrowheads) running parallel to MTs. Scale bars, 50 nm. (I, J, K) Tomographic slices with actin singlet filaments interacting with MTs. The interactions are marked in cyan. Scale bar in (I), 25 nm; scale bars in (J, K), 50 nm.