Discovery and in vivo characterization of novel TOG domain-containing proteins using C. elegans

This study identifies and characterizes two novel, reduced-domain TOG proteins (TOD-1 and TOD-2) in *C. elegans* that regulate tubulin dynamics to ensure proper sperm localization and function, challenging the prevailing view that amoeboid sperm motility is microtubule-independent.

The Gist

The Big Picture: Finding New "Construction Managers" in a Tiny Worm

Imagine the inside of a cell as a bustling construction site. To keep the building standing and moving, you need strong scaffolding. In cells, this scaffolding is made of microtubules—tiny, hollow tubes that act as highways for transporting cargo and giving the cell its shape.

To build and maintain these highways, cells use special "construction managers" called TOG domain proteins. These managers grab onto the raw building blocks (tubulin bricks) and help snap them together into long, sturdy tracks.

For a long time, scientists knew about the famous managers in this family (like the XMAP215 team). But in this study, researchers at the University of North Carolina looked at the tiny roundworm, C. elegans, and discovered two brand new, previously unknown managers they named TOD-1 and TOD-2.

The Discovery: The "Oddballs" of the Family

Most construction managers in the TOG family are like a team of four or five people working together, holding onto the building blocks with multiple hands (called "TOG domains").

• TOD-1 is unique because it's a "lone wolf." It only has one hand (one TOG domain) instead of the usual four or five. It's like a construction foreman who usually needs a crew, but this one is trying to do the job solo.
• TOD-2 is a bit more standard, having two hands, but it's still a new character on the scene.

Using computer modeling (like a 3D blueprint generator called AlphaFold), the scientists predicted that these new managers are very picky. Unlike their famous cousins who help build the long highway tracks, TOD-1 and TOD-2 seem to only grab onto the loose, individual bricks floating around, not the finished highway itself.

The Mystery: Why Do the Worms Lay "Empty" Eggs?

The researchers decided to see what happens if they remove these new managers from the worms. They used gene-editing tools (CRISPR) to delete the instructions for making TOD-1 and TOD-2.

The Result: The worms looked mostly normal. They grew up, moved around, and had babies. However, there was a strange glitch: the female worms started laying unfertilized eggs.

Think of it like a bakery that keeps baking bread but forgets to put the yeast in. The bread (eggs) comes out, but it's empty and useless. In the wild, this is a waste of energy.

The Real Culprit: A Sperm Navigation Problem

Why were the eggs empty? The researchers suspected it had to do with the sperm.

In C. elegans, sperm are tiny, amoeba-like blobs that crawl to find the egg. Scientists used to think sperm didn't need microtubules (the highways) to move; they just used a different muscle protein. But this study suggests that TOD-1 and TOD-2 are actually crucial for sperm navigation.

Here is the analogy:
Imagine the sperm as a delivery driver trying to get a package (the sperm nucleus) to a specific drop-off point (the spermatheca, where the egg waits).

1. The Glitch: In worms without TOD-1 or TOD-2, the delivery drivers get lost. They crawl into the wrong room (the uterus) and can't find their way back to the drop-off point.
2. The Consequence: The egg is waiting at the drop-off point, but the driver never arrives. The egg gets laid anyway, but since no sperm made it there, it's an empty, unfertilized egg.
3. The Timing: This problem gets worse as the worm gets older. It's like a GPS system that works okay for the first few deliveries but starts to fail as the battery drains.

The Twist: It's Not Just the Sperm

To prove it was the sperm's fault and not the egg's, the researchers did a "dating experiment." They took normal female worms and mated them with mutant male worms (who lacked TOD-1/2).

The result? The normal females still laid a few empty eggs when mated with the mutant males. This confirmed that the sperm were the ones having trouble navigating. However, the defect wasn't 100% sperm-based; the researchers suspect these proteins might also play a subtle role in the female's reproductive tract, acting as a "welcome mat" or a signal flare that helps the sperm find their way home.

Why Does This Matter?

1. New Rules for Biology: We thought we knew how the "construction managers" (TOG proteins) worked. Finding one that only has one hand (TOD-1) and acts differently than the big teams breaks the old rules. It suggests nature has many more ways to build and regulate cell structures than we thought.
2. Sperm are More Complex: We used to think sperm were simple, microtubule-free blobs. This study opens a new door: maybe sperm do use microtubules for navigation, just in a very subtle, specialized way that we haven't seen before.
3. Fertility Clues: Understanding how sperm find their way to the egg in worms could eventually help us understand similar navigation failures in human fertility issues.

In a Nutshell

The scientists found two new, weirdly shaped "construction managers" in a tiny worm. When they removed these managers, the worms' sperm got lost on their way to the egg, resulting in a lot of empty eggs being laid. This discovery changes how we think about sperm movement and proves that even the simplest cells have complex, hidden systems for getting things done.

Technical Summary

1. Problem Statement

Microtubule dynamics are essential for cellular architecture, division, and transport, regulated by a complex network of microtubule-associated proteins (MAPs). While the TOG (Tumor Overexpressed Gene) domain superfamily is well-known for regulating microtubule polymerization (specifically the XMAP215, CLASP, and Crescerin families), the full repertoire of these proteins in model organisms like Caenorhabditis elegans was not fully characterized.

Specifically, the study addresses:

• The existence of uncharacterized TOG domain-containing proteins in the C. elegans proteome.
• The functional role of these proteins, particularly given the unique biology of C. elegans sperm, which was historically considered microtubule-independent (relying on Major Sperm Protein for amoeboid motility).
• How variations in TOG domain structure (e.g., reduced numbers of domains or divergent sequences) affect protein function and organismal fertility.

2. Methodology

The researchers employed a combination of bioinformatics, structural modeling, and in vivo genetic manipulation:

• Bioinformatic Screening: They searched the C. elegans InterPro database for TOG domains (IPR034085), identifying two novel genes: T08D2.8 and F54A3.2.
• Phylogenetic Analysis: Maximum Likelihood Trees were constructed using MUSCLE alignment and the Jones-Taylor-Thornton model to classify the novel proteins against known XMAP215, CLASP, and Crescerin families across five species.
• Structural Modeling: AlphaFold3 was used to predict protein structures. The models were threaded through the S. cerevisiae Stu2 TOG2 domain structure (PDB: 4FFB) using ChimeraX to predict binding interfaces with tubulin dimers (kinked vs. straight conformations).
• Genetic Engineering (CRISPR/Cas9):
  • Knockouts: Open reading frames (ORFs) of tod-1 and tod-2 were replaced with mNeonGreen (creating transcriptional reporters and null mutants simultaneously).
  • Tagging: Endogenous C-terminal tagging with mNeonGreen was performed to visualize protein localization.
  • Strains: Wild-type N2, tod-1Δ, tod-2Δ, and fog-2(q71) (female-only) strains were used.
• Phenotypic Assays:
  • Brood Size & Viability: Counting hatched larvae vs. dead embryos to assess fertility and embryonic viability.
  • Unfertilized Oocyte Count: Visualizing and counting dark, circular oocytes lacking eggshells on culture plates.
  • Sperm Positioning: Using mCherry::his-58 to label sperm nuclei, researchers quantified sperm distribution in the proximal germline, spermatheca, and uterus at 24h and 48h post-L4 stage.
  • Mating Assays: Crossing tod mutant males with fog-2 females to isolate the contribution of sperm function versus somatic/oogenic defects.
• Imaging: Confocal microscopy (Nikon A1R) and widefield microscopy (Nikon Eclipse Ti2-FP) were used for live imaging and DIC analysis.

3. Key Contributions

• Discovery of Novel Proteins: Identification and naming of two previously uncharacterized TOG domain proteins: TOD-1 (one TOG domain) and TOD-2 (two TOG domains).
• Structural Insight: Demonstration that these proteins belong to the XMAP215 family but possess unique structural features:
  • TOD-1 contains only one TOG domain (unusual for animals) with only five HEAT repeats (typically six), suggesting a truncated binding interface.
  • Both proteins are predicted to bind soluble tubulin dimers rather than the microtubule lattice, based on AlphaFold modeling and alignment with the "kinked" tubulin conformation.
• Functional Characterization: Establishing that these proteins are critical for sperm localization and function in C. elegans, challenging the dogma that C. elegans sperm motility is entirely microtubule-independent.

4. Key Results

• Expression Patterns: Both TOD-1 and TOD-2 are expressed in the germline and sperm. TOD-2 is more abundant than TOD-1.
• Viability and Development: tod-1 and tod-2 single knockouts are viable and generally healthy. There were no significant changes in total brood size or embryonic viability compared to wild-type.
• Unfertilized Oocyte Phenotype:
  • Knockout hermaphrodites laid a significantly increased number of unfertilized oocytes.
  • This phenotype was age-onset, appearing earlier and more frequently than in wild-type animals.
• Sperm Defects:
  • Cellularization: Mutants showed an accumulation of cellularized oocytes in the proximal germline, indicating a failure in fertilization.
  • Sperm Homing: At 24 hours post-L4, tod mutants had a significantly lower fraction of sperm localized in the spermatheca and proximal germline compared to wild-type. By 48 hours, sperm positioning normalized, suggesting a transient defect in sperm homing/migration.
• Sperm-Specific Role:
  • Mating fog-2 females with tod mutant males resulted in normal brood sizes and viability, but an increase in unfertilized oocytes.
  • This confirms that the defect lies primarily in sperm function (specifically the ability to return to the spermatheca after displacement) rather than in the oogenic germline or somatic tissues.

5. Significance

• Redefining Sperm Biology: The study provides the first evidence that microtubule regulatory proteins (TOG domains) are required for C. elegans sperm function. While C. elegans sperm motility is driven by Major Sperm Protein (MSP), these results suggest that residual tubulin or microtubule dynamics play a crucial, previously overlooked role in sperm localization and homing to the spermatheca.
• Expanding TOG Domain Rules: The discovery of TOD-1, a protein with a single, truncated TOG domain (5 HEAT repeats), challenges existing models of how XMAP215 family proteins function. It suggests that the "rules" for tubulin binding and polymerase activity are more flexible than previously thought.
• Mechanism of Fertility: The findings link the regulation of free tubulin dimers (via TOD-1/2) to the successful retention of sperm in the spermatheca, a critical step for fertilization. This opens new avenues for understanding how cytoskeletal regulators coordinate cell-cell interactions in the reproductive tract.
• Future Directions: The paper sets the stage for investigating how truncated TOG domains regulate microtubule dynamics and whether these proteins interact with other sperm-enriched tubulin binders (like Cylicin) to scaffold organelles or regulate residual microtubules.