Microtubule dynamic is tightly regulated by assembly-promoting and destabilizing factors, as it takes an essential role in cellular processes, such as mitosis and intracellular transport. In this paper, Goodwin and her group hypothesize that Drosophila Patronin has a regulatory role at the microtubule (MT) minus end. This hypothesis is based on a previous genomic screen that associated spindle morphology defect to the loss of Patronin; and the observation of its human homolog at the minus end. The remarkable stability of free MT minus end has been reported, however the mechanism is yet elucidated. As the majority of research has focused on the plus end, this paper is highly significant by identifying Patronin as a protecting factor at the minus end competing against depolymerizing protein, Kinesin-13.
To approach the hypothesis, Goodwin and her group derived two major experimental procedures: (i) photobleaching, and (ii) in vitro assays. First, photobleaching was used to look at the movement of MT. It created a “dark box” on a region on the MT to identify whether the MT is either transported by motor protein or treadmilling. The bleach mark would be stationary if transported, whereas it would move toward the minus end if treadmilling. Furthermore, several in vitro assays were performed. Notably, the anchoring assay was performed by attaching GFP-Patronin to Anti-GFP coated coverslip, and subsequently observing the rhodamine-labeled microtubules from one end. This anchoring assay allowed the group to identify which MT end is anchored to GFP-Patronin. For the gliding assays, kinesin or dynein was added after microtubule anchoring. As kinesin moved toward the plus end and dynein moved toward the minus end, the binding selectivity of Patronin can be determined.
By comparing to RNAi control, the group found that most Patronin-depleted Drosophila S2 cells have a lowered MT density and an increase of free MT during interphase. These free MT were released from nucleating sites and treadmill across cytoplasm to the cell periphery. The group continued the investigation to explain the increased depolymerization.
The codepletion of Kinesin-13 member (shown in figure), KLP10A, and Patronin, reversed the Patronin-depleted phenotype. During metaphase, the codepletion achieved longer pole-to-pole metaphase spindle than control, and decreased poleward flux of tubulin subunits. Interestingly, Patronin-depleted cells displayed two distinct types of bipolar spindle: normal form that aligns with metaphase plate; and collapsed form that resemble monopolar spindle. From the domain analysis of Patronin, the CC domain was localized with small MT nucleating foci, and the CKK domain was localized along MT. From the gliding assays, Patronin was found to bind to MT at the minus end. In the presenceof Patronin and Kinesin-13, Patronin, MT depolymerization was only shown at the plus end but not at the minus end. Increasing KLP10A homolog, MCAK, also correlated with higher MT depolymerization at minus end when the concentration of Patronin remains constant. In conclusion, Patronin protected the MT minus end against Kinesin-13-mediated depolymerization.
Goodwin and her group have presented a convincing story of the protective function of Patronin. Different domains of Patronin are localized in distinctive regions, suggesting domains might work cooperatively. CC domain of EB1 is necessary for EB1 to bind to MT. Here, CKK alone is able to direct Patronin to localize along MT. Why is there a redundant role of two different domains in localizing Patronin to MT? The CKK and CH domains may be required to target to the minus end. The CKK and CH domains may need to interact to uncover buried residues for the minus end targeting. To approach the hypothesis, we should align different Patronin homologs. Then, screen out the potential conserved regions in CKK and CH domains. Next, mutate charged residue to alanine residue. If the region is the sites required for minus end specificity, alanine mutants should not localize to the minus end of MT. These results would help us to understand how Patronin obtains its MT minus end specificity.
Goodwin SS, Vale RD. Patronin regulates the microtubule network by protecting microtubule minus ends. Cell. 2010 October 15; 143(2): 263-274.