Project Description
Plastin, an actin cross-linking protein, has been shown to be instrumental for efficient cytokinesis and polarization in C. elegans zygotes. By increasing connectivity within the cortical actomyosin network, plastin provides strength to the network, to enable a coordinated cortical flow required to segregate proteins during zygote polarization. Similarly, it facilitates the assembly of contractile foci during cytokinesis. With plastin connecting and bundling actin filaments together, long-range coordinated contractility of the actomyosin network is possible. Without it, key processes of early embryogenesis are delayed, and at times, fail.
These findings were published in J Cell Biol.
Ding, WY et al., Plastin increases cortical connectivity to facilitate robust polarization and timely cytokinesis, J. Cell Biol. vol. 216 no. 5 1371-1386, doi: 10.1083/jcb.201603070)
More information on the Zaidel-Bar Lab.
Figure: Plastin (green) and non-muscle myosin II (red) in the cortex of a newly fertilized C. elegans zygote during polarization (top panel) and cytokinesis (bottom panel) using spinning disk confocal microscopy. Co-localization between green and red signals signifies localized cortical contractility.
Plastin, an actin cross-linking protein, has been shown here to be instrumental for efficient cytokinesis and polarization in C. elegans zygotes.
By increasing connectivity within the cortical actomyosin network, plastin provides strength to the network, to enable a coordinated cortical flow required to segregate proteins during zygote polarization. Similarly, it facilitates the assembly of contractile foci during cytokinesis. With plastin connecting and bundling actin filaments together, long-range coordinated contractility of the actomyosin network is possible. Without it, key processes of early embryogenesis are delayed, and at times, fail.
Understanding the basics
Crosslinking of actin filaments is facilitated by actin binding proteins such as a-actinin, fascin or filamin. These proteins tether actin filaments together to strengthen the cytoskeleton, and enable their arrangement into higher order actin-based structures. In filopodia, crosslinking of actin filaments provides the rigidity needed to overcome the compressive force of the plasma membrane, which individual actin filaments otherwise lack. Filopodia in nerve growth cones contain tightly packed bundles of actin filaments that usually contain more than 15 parallel filaments. These are likely to be oriented with their barbed ends towards the tip. Mechanically, a crosslinked filopodial bundle functions as an effective elastic rod. Bundle stiffness increases with the number of bundled filaments and so contributes to the overall filopodium length. In lamellipodia, crosslinking also strengthens the actin filaments, however in this case the filaments form a branched network, which is connected at certain points to membrane bound proteins and focal adhesions (as depicted in Fig 1). Crosslinking also increases the ATPase activity of myosins and increases the tension on actin filaments.
Read more on actin cross-linking
Cell polarity refers to the intrinsic asymmetry observed in cells, either in their shape, structure, or organization of cellular components. Most epithelial cells, migrating cells and developing cells require some form of cell polarity for their function. These cells receive information about their surroundings via extracellular biochemical and mechanical cues and translate those information into polarity of the plasma membrane, its associated proteins and cytoskeletal organization. Once established, cell polarity is maintained by transcytosis, in which vesicles carry incorrectly-localized membrane proteins to the correct regions in the plasma membrane. In addition, tight junctions, which act as ‘fences’ against transmembrane diffusion, lock the asymmetry in place. Therefore, mechanobiology plays an essential regulatory role in both the establishment and maintenance of cell polarity.
Epithelial cells establish an apical-basal polarity, which results from the differential distribution of phospholipids, protein complexes, and cytoskeletal components between the various plasma membrane domains, reflecting their specialized functions. The membrane facing the lumen or free surface is known as the apical membrane, while the membrane oriented away from the lumen, contacting the extracellular matrix, is known as the basal membrane and the sides of the cell contacting the neighboring cells form the lateral membrane. The apico-basal polarization of epithelial cells is known to be a pre-requisite for their fundamental biological roles. These include regulating the vectorial transport of ions across cell sheets during their barrier function as well as ensuring directionality during their secretory and absorptive functions.
In other specialized cells such as immune cells and neurons, cell polarity enables the short-range and long-range transmission of various electrical and biochemical signals. For instance, A typical unipolar neuron has a highly distinctive shape and structure, with one end adapted to receive signals through highly branched dendrites. This signal is then transmitted down an axon, which can stretch the length of the body. At the other end of the cell is the axon terminal, where the synapses are located. These synapses can release chemical neurotransmitters in order to propagate the signal or effect an action such as muscle contraction.
Read more on cell polarity
The Study in Detail
Key Findings
- Previous findings in other in vitro systems had found that a-actinin (refs) was crucial for in the establishment of cortical contractility. In the elegans zygote however, a-actinin did not contribute to the division or polarization of the zygote. Instead, plastin was key to these processes.
- Plastin PLST-1 increases cortex stiffness. When plastin function was disrupted through the expression of a loss-of-function mutant, membrane invaginations were observed in the posterior cortex. This is because the cortex was unable to resist being pulled on by microtubules when plastin was absent. As the microtubules pulled, the weakened cortex gave way, and invaginations in the membrane occurred.
- Plst-1 expressing zygotes displayed erratic cortical flows that weakened after 90 seconds. This was in contrast to the well-ordered and consistent flows observed in control zygotes. These differences indicated that connectivity within the actomyosin network was necessary for coordination of the cortical flow.
- Consistent with disrupted cortical actomyosin flows, the segregation of the anterior and posterior PAR proteins (PAR2 and PAR6) were disrupted in plst-1 zygotes. Here, two phenotypes were evident, one mild and the other severe. In the latter, polarity was not rescued.
- Expression of truncated plastin did not affect the recruitment of NYM-2 to the cortex, but it did prevent the merging of NYM-2 proteins into the large foci that were observed in control zygotes. In plst-1 zygotes, NYM-2 remained scattered throughout the cortex.
- In most plst-1 zygotes, cytokinesis was delayed, but was eventually completed. This delay was attributed to disrupted initiation of the cleavage furrow. Of the 15% of plst-1 zygotes that did not complete cytokinesis, defective furrow ingression was noted. Delayed cytokinesis in plst-1 zygotes was attributed to disrupted recruitment of non-muscle myosin-2 (NMY-2) to the cytokinetic ring.
- Simulations using Cytosim highlighted that the optimal level of contractility occurs when cross-linkers are present in the system at a ratio of 2:1 (motor protein to cross-linker). Increasing or decreasing the levels of cross-linkers reduced contractility significantly.
Methods and Controls used in the study
- Assessed the function of a truncated PLST-1 protein (370bp deletion of the second-to-last exon, which encodes the third, and most of the forth, CH domain).
- Zygotes expressing plst-1 (tm4255) (which produced a truncated protein with a loss-of-function) were compared to zygotes expressing wild-type plst-1::GFP .
- Zygotes expressing the truncated PLST-1 were imaged with confocal microscopy
- PLST-1 was visualized by a translational fusion of PLST-1 with GFP, using CRISPR/Cas9
- Early embryogenesis was monitored by co-expression of a membrane marker (GFP::PLC1δ-PH), and a histone marker, (HIS-58::mCherry)
- Microtubule pulling was monitored using GFP::tubulin and PH::mCherry.
- Contractility was simulated using Cytosim (Ref), with filaments given the flexibility of F-actin filaments with varying levels of motor and cross-linking proteins added.
- Other methods used include cortical laser ablation, particle image velocimetry analysis,
