Uncropped SDS-PAGE gel used in Fig 4C.

During host cell invasion, microsporidian spores translocate their entire cytoplasmic content through a thin, hollow superstructure known as the polar tube. To achieve this, the polar tube transitions from a compact spring-like state inside the environmental spore to a long needle-like tube capable of long-range sporoplasm delivery. The unique mechanical properties of the building blocks of the polar tube allow for an explosive transition from compact to extended state and support the rapid cargo translocation process. The molecular and structural factors enabling this ultrafast process and the structural changes during cargo delivery are unknown. Here, we employ light microscopy and in situ cryo-electron tomography to visualize multiple ultrastructural states of the Vairimorpha necatrix polar tube, allowing us to evaluate the kinetics of its germination and characterize the underlying morphological transitions. We describe a cargo-filled state with a unique ordered arrangement of microsporidian ribosomes, which cluster along the thin tube wall, and an empty post-translocation state with a reduced diameter but a thicker wall. Together with a proteomic analysis of endogenously affinity-purified polar tubes, our work provides comprehensive data on the infection apparatus of microsporidia and uncovers new aspects of ribosome regulation and transport.</div

Subtomogram averaging workflow followed using the Dynamo package.

(a) Schematic workflow of the subtomogram averaging procedure to generate the ribosome volume. (b) Two 60°-related views of ribosome reconstruction (transparent white), fitted with the structure of the V. necatrix ribosome (PDB ID: 6RM3, magenta). (c) Schematic workflow of the subtomogram averaging procedure used to create the reconstructions of the segments of polar tube outer layers. The scheme is shown for cargo-filled tubes, and a similar methodology was used for empty tubes. (d) Subtomogram averaging workflow used to reconstruct dimeric ribosomes from clustered particles in sporoplasm-filled tubes. (e) Low-pass filtered subtomogram averages of ribosome dimers placed back into their original location in germinated polar tubes. Averages are shown in yellow, and the central slice of the tomogram slice is shown in gray. (PNG)</p

Mass spectrometry results of a PTP3 affinity-purified sample.

The list of mass spectrometry hits is arranged according to peptide-spectrum match (PSM). The number of consecutive histidines (3xHis, 4xHis, 7xHis) in the protein is listed. PTP3 is the only protein with 7 consecutive histidines. (PDF)</p

In-gel mass spectrometry analysis of PTP3 affinity-purified sample.

The 2 prominent protein bands indicated by an asterisk (*) in Fig 4C, one around 130 kDa and 20 kDa were sliced and analyzed. The list of mass spectrometry hits is arranged according to peptide-spectrum match (PSM). (PDF)</p

Tracking polar tube eversion to understand germination dynamics and tube length.

(a) A kymograph obtained via live light microscopy analysis of polar tube firing events from Vairimorpha necatrix. The spore at the bottom of the kymograph is denoted by “S” and the sporoplasm ejected on the distal end is indicated as “SP.” (b) Length over time diagrams of all analyzed polar tube eversion events. The average length over time is colored in green. (c) Bar plots of polar tube maximal length (yellow), length at the end (blue), and maximum velocity distribution (gray). The raw data used to create the plots can be found in the S1 Data. (PNG)</p

Tomographic volume of a ribosome-filled-germinated polar tube overlaid with the corresponding segmentation.

Tomographic volume of a ribosome-filled-germinated polar tube overlaid with the corresponding segmentation.</p

Endogenous PTP3 pulldown.

(a) Representation of the 1,354 amino acids long V. necatrix PTP3. Marked are the N-terminal predicted signal peptide (highlighted in gray), the histidine stretch (red area zoomed in with sequence) that was used for purification, and the disorder prediction (black line). (b) Schematic representation of the endogenous purification of PTP3. (c) The different purification steps shown in (b) have been analyzed on an SDS PAGE. See the S1 Raw Image file for the uncropped gel. The 2 bands in the “Elution” lane (indicated by the red stars) were excised for in-gel mass spectrometric analysis. (d) A pie chart showing the distribution of all mass spec hits detected in the elution sample, shown in (c), classified using functional and structural annotation of the V. necatrix genome (manuscript in preparation). (e) Mass spec hits from the classes: PTPs, RBLs, and proteins of unknown function, are sorted based on the number of significantly detected unique peptides (high to low). Gene IDs are presented with colors for the respective classes, along with names for the encoded proteins, molecular weights, and structurally disordered regions predicted using PrDos [75]. The white symbol in front of the gene ID indicates close genomic localization. The full mass spectrometry data table can be found in S2 and S3 Tables.</p

Visualizing polar tube wall features from cargo-filled and empty tubes.

(a) Central slices through representative cryo-tomograms from cargo-filled and empty polar tubes. Segmentations of the inner and outer layers are superimposed onto 1 side of the tube in both tomogram slices. The scale bar is 100 nm. (b) Zoomed in sections of the tube wall from (a). The scale bar is 10 nm. (c) A plot depicting the thickness of each outer protein layer and membrane bilayer, as measured across various tomograms, for cargo-filled and empty polar tubes. The p-values of two-tailed, unpaired Student’s t test analyses are shown above the compared plots. The raw data underlying this figure can be found in S1 Data. (d) Side-by-side comparison of 2 slab views of subtomogram averages obtained from cargo-filled (left) or empty (right) polar tube walls. The volumes were segmented and colored by membrane and outer layer. The views at the top (slabs along the polar tube) are shown below rotated 90° around the Y-axis (slabs across the polar tube). Map regions are colored as in (a). (e) A schematic representation of polar tube and outer protein layer remodeling during cargo movement.</p

Raw data points used in the main manuscript figures are organized in individual sheets labeled according to the corresponding figure.

Raw data points used in the main manuscript figures are organized in individual sheets labeled according to the corresponding figure.</p

Representative tomograms depicting empty or sporoplasm-packed germinated polar tubes.

(a) A schematic representation of the methodology for on-grid freezing and collecting tomograms of germinated polar tubes. (b) A graph representing the internal diameter of polar tubes (PTempty and PTcargo) visualized using cryo-ET. Each dot represents 1 tube, and the line represents the mean diameter. The raw data underlying this figure can be found in the S1 Data file. (c) Representative tomograms of PTempty or polar tubes filled with electron-lucent material or completely devoid of cellular cargo. The central section of a tomogram is shown with regions of interest indicated with arrows (magenta for the outer wall, pink for the lipid bilayer, and blue for vesicles). (d–f) Representative tomograms from PTcargo or polar tubes filled with cellular cargo where (e and f) contained ribosome spirals inside tubes. The central section of a tomogram is presented, and regions of interest are indicated with arrows (light blue for the outer tube wall, pink for the lipid bilayer, and yellow for ribosomes). For (e and f), additional views corresponding to the boxed regions and corresponding segmented tomograms are also presented. (PNG)</p