SPOTLIGHT ARTICLES: Patronin regulates the microtubule network (Goodwin et al. 2010) REVIEW

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.

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Goodwin SS, Vale RD. Patronin regulates the microtubule network by protecting microtubule minus ends. Cell. 2010 October 15; 143(2): 263-274.

A visit to the current controversy: manmade mutant H5N1 influenza virus (2)

From the last issue, a current controversy about the mutant H5N1 was discussed. I will continue the discussion by talking about some essential aspects of deriving successful mutants. These mutants can have completely different tropism for tissues or animals. The understanding by how a mutant is derived had recently stirred a debate between academic freedom and bioterrorism. Personally, I think the understanding of how to derive mutants is crucial in aiding us to define how new viruses are evolved.

Receptor influences viral entry

Binding to the cellular receptor is the first step when the virus comes in contact with the host cell. The cells that express a particular type of receptor make them susceptible to viral infection. Other than the susceptibility, one must also consider if cells have the required machinery for a productive infectious cycle. This is what defined the term, permissive. Here, I will discuss a paper from Higgs’ lab at the University of Texas, called a single mutation in Chikungunya (CHIKV) virus affects vector specificity and epidemic potential (shown right).

The CHIKV strain that circulates in 2005-2006 epidemic on Reunion island is different from normal CHIKV infection. This particular strain is not transmitted through the normal route: Aedes aegypti (which is the same species responsible for dengue viral infection in human). In fact, it is transmitted by the Asian tiger mosquito called Ae. albopictus. By sequencing, these researchers found a particular mutation at the E1 protein at the site 226 (mutated from Alanine to Valine).

Interestingly, when they look at the replication of both strains within the same organism, they found that the 226V mutant is more successful in replicating within the Ae. albopictus but not at the Ae. aegypti.

They also supported this result by looking at the dissemination of each strain. It is clear that the 226V mutant is more successful in completing the infectious cycle against 226A mutant only in Ae. albopictus. In contrast, 226V mutant does not have an advantage in Ae. aegypti. The researchers then went on to support the conclusion by infecting animals with the mosquitoes.

E1 is a part of the spike on the CHIKV envelope. Complexed with E2 in the hetero-trimeric spike structure, this complex facilitates the interaction with cellular receptor, entry, and budding. The mutation in E1 protein can completely change the tropism for its vector. It is also able to compete against wildtype viruses. The bottom line is the change in viral envelope proteins can change its preference of cells that it can infect. In other words, envelope proteins must be mutated in a way that it can recognize cells that express different receptors.


PLoS Pathog. 2007 Dec;3(12):e201.

A single mutation in chikungunya virus affects vector specificity and epidemic potential.

Tsetsarkin KA, Vanlandingham DL, McGee CE, Higgs S.

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Lab Protocol #1: Making Agar Plate, LB Broth

Making Agar Plate
20g LB Broth
1L H2O
15g Bacto Agar
Mix well first. Pour the mixture into a 2L flask.

Some bacto agar will not be completely dissolved. Therefore, you can always let the bacto agar to settle in your 2L flask, then take the top phase of liquid to flush the remaining bacto agar out.

After that, put it into autoclave machine (wet cycle: this is often preset. But usually it is 100C for 20-30 minutes)

If you wish to make plates for penicillin, wait until the flask to cool down a bit, don’t wait for too long, otherwise the agar will solidify (if you can bear the temperature with your bare skin, the temperature is good). At this time, add penicillin at 1:1000 dilution.

Making LB Broth

40 g LB Broth

2L H2O

Mix well, pour into 4 glass bottles, then autoclave

*When autoclaving liquid in glass bottles, make sure to not tighten the glass bottle completely.