Developmental Biology - Branching|
Microtubules Branch To Spur Mitotis
A first in animals, seeing the essential mitotic split made in living cells...
Branching nucleation is a fundamental and conserved (between species) process. It is one of the essential steps in mitosis - the equal division of a single cell into two cells. But, it is difficult to capture in model animal systems, according to cell biologist Thomas Maresca, University of Massachusetts Amherst.
"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."
Thomas J. Maresca PhD,
Biology, University of Massachusetts; Molecular and Cellular Biology Graduate Program, University of Massachusetts, Amherst, Massachusetts, USA.
Maresca and senior research fellow Vikash Verma PhD,
Biology Department, University of Massachusetts, Amherst, have for the first time observed and recorded in animal cells - a pathway called branching microtubule nucleation. This mechanism of cell division has been imaged in cell extracts and plant cells but not clearly observed in animal cells. The work appears this month in the Journal of Cell Biology.
Supported by NIH's National Institute of General Medical Sciences, the scientists set out to explore what Vikash Verma calls "rules of faithful and complete division" in fruit fly cells, in order to understand how structures called microtubules help define where a cell will split during the mitosis process.
"This has been studied since microscopy made it possible to see cells divide; 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? The ultimate conclusion of mitosis, the actual division of one cell into two cells."
Thomas J. Maresca PhD
In normal cell division, chromosomes line up near the center of the cell where a structure called the spindle aligns copies of each chromosome using a bridge-like structure called the kinetochore. When all the chromosomes are aligned, microtubules pull chromosome copies apart much like a zipper. The cell then continues dividing the segregated chromosomes and produces two daughter cells. Each with a complete copy of the genome. While imaging microtubules, often described as nano-scale highways, biologists noticed the spatial cue for locating the division plane requires microtubules.
"Microtubules grow and touch the edges inside the cell membrane. These 'plus-ends' tell the cell where to divide - kicking into gear and recruiting regulatory proteins to assemble into a ring that will constrict and split one large cell into two smaller ones, acting much like a purse-string.
It seems that all microtubule tips have this special ability to trigger a purse-string pathway. But over time, something changes and only the tips in the middle of the cell retain that ability. We found what we think is a very important spatial cue for how a cell positions its division plane."
Thomas J. Maresca PhD
Visualizing the behavior of microtubules during cell division in detail is typically hampered by the fact that so many are growing and shrinking at the same time.
Verma: "It looks like many highways converging at the same place and time in the spindle — like a Los Angeles freeway map." By using a powerful technique called total internal reflection fluorescence (TIRF) microscopy, Verma could more easily visualize the properties of individual microtubules causing Maresca to add: "We went from a stressful LA traffic jam to a 'Sunday drive on a country road.'
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:"We knew we could 'tag' proteins that regulate the process with different colors and further quantify fundamental parameters of the phenomenon. All of a sudden we realized this is the first time anyone could see this happening in living animals cells."
Centrosome-mediated microtubule (MT) nucleation has been well characterized; however, numerous noncentrosomal MT nucleation mechanisms exist. The branching MT nucleation pathway envisages that the a-tubulin ring complex (a-TuRC) is recruited to MTs by the augmin complex to initiate nucleation of new MTs. While the pathway is well conserved at a molecular and functional level, branching MT nucleation by core constituents has never been directly observed in animal cells. Here, multicolor TIRF microscopy was applied to visualize and quantitatively define the entire process of branching MT nucleation in dividing Drosophila cells during anaphase. The steps of a stereotypical branching nucleation event entailed augmin binding to a mother MT and recruitment of a-TuRC after 15 s, followed by nucleation 16 s later of a daughter MT at a 36° branch angle. Daughters typically remained attached throughout their ~40-s lifetime unless the mother depolymerized past the branch point. Assembly of branched MT arrays, which did not require Drosophila TPX2, enhanced localized RhoA activation during cytokinesis.
Vikash Verma, Thomas J. Maresca.
This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).
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Image from movie
available online. In the fruit fly brain, branched microtubule (MT) arrays increase the activity of RhoA near the fruit fly brain cortex. Time-lapse microscopy of an anaphase cycle in a fruit fly S2 cell shows co-expression of markers for active RhoA or Ras homolog gene family, member A. Enriched Rhotekin signals were seen near the branched MT arrays. A nearby region without MT plus, doesn't show increased Rhotekin. UPPER LEFT: Tag-RFP-T-a-tubulin. UPPER RIGHT: Rhotekin-EGFP LOWER LEFT: Merged image. CREDIT Vikash Verma, Thomas J. Maresca.