How microtubules branch in new directions, a first look in animals

In this work supported by NIH's National Institute of General Medical Sciences, the researchers set out to explore specific mechanics of cell division, what Verma calls "the rules of faithful and complete division," in fruit fly cells. In particular, they want to understand how structures called microtubules help to define where the cell splits in half during the division process.

Maresca explains, "This has been studied for a long time, since microscopy made it possible to see cells divide, but very intensely for 40 or 50 years. What are the cues that tell a cell where to divide? How does the cell know where to put the division plane? It's the ultimate conclusion of mitosis, the actual division of the cell into two."

In normal cell division, chromosomes line up near the center of the cell, where a structure called the spindle aligns copies of each chromosome by interacting with a bridge-like structure called the kinetochore. When all the chromosomes have been aligned, microtubules pull the chromosome copies apart like a zipper. The cell then physically divides at a location positioned between the segregated chromosomes to produce two daughter cells, each with a complete copy of the genome.

In imaging the microtubules, often described as nano-scale highways, the biologists noticed that the spatial cue for locating the division plane requires microtubules, Maresca says. "They grow out to touch the edges inside the cell membrane. Vikash found that the growing tips of the tubes, the 'plus-ends' that touch the membrane, say to the cell, 'This is where to divide.' Regulatory proteins get recruited to the site contacted by the plus-ends kicking into gear and a whole new pathway assembles a ring that will constrict like a purse string to split one large cell into two smaller ones."

Timing plays a role, as well, the researchers found. "It seems that all the microtubule tips have the special ability to trigger the purse-string pathway," Maresca says, "but over time, something changes and only the tips in the middle of the cell retain that ability." Referring to work published in eLife in February, he adds, "We found what we think is a very important spatial cue for how the cell positions its division plane."

Visualizing the behavior of microtubules during cell division in detail is typically hampered by the fact that so many microtubules are growing and shrinking at the same time throughout the cell, Verma says. "It's like many highways converging at the same place and time in the spindle. It looks like a Los Angeles freeway map." But by using a powerful technique called total internal reflection fluorescence (TIRF) microscopy, Verma could more easily visualize the dynamic properties of individual microtubules. Maresca adds, "We went from a stressful L.A. traffic jam into a Sunday-drive-on-a-country-road view."

That is when they witnessed the branching. Using multi-color TIRF microscopy, the researchers could now clearly see and quantitatively define the branching microtubule nucleation process. To the best of their knowledge, this had never been visualized before in real time in animal cells. "It was very exciting," Verma recalls.

Maresca says, "When you see such beautiful things right before your eyes, you just have to follow it. This project started out as an investigation of how cells define where they divide, but we saw this branching phenomenon so often and so clearly that we realized we had to look at it more closely. We don't think you could have seen the branching process as well in other model systems as you can in our fruit fly cells. It highlights the fact that every model system has its strengths and its weaknesses and, in this case, our cells and the phase at which we were imaging them just offered a uniquely beautiful, birds-eye view of branching. We could actually see all of this happening in real time before our eyes."

Once they could visualize the entire process of branching nucleation in a cell, he adds, "We knew we could next 'tag' proteins that regulate the process with different colors to further quantify fundamental parameters of the phenomenon. All of a sudden we realized that this is the first time one could see this happening in living animals cells."

Branching nucleation is fundamental and conserved, one of the essential parts of mitosis, but it's been difficult to directly visualize in other model systems, Maresca points out. "The course of this project was a reminder that some of the most exciting work we do as scientists is unplanned and, especially for microscopists, begins with seeing something in the cell unfold right before your eyes."