Margaret A. Titus

Myosin I Contributes to the Generation of Resting Cortical Tension

The amoeboid myosin Is are required for cellular cortical functions such as pseudopod formation and macropinocytosis, as demonstrated by the finding that Dictyostelium cells overexpressing or lacking one or more of these actin-based motors are defective in these processes. Defects in these processes are concomitant with changes in the actin-filled cortex of various Dictyostelium myosin I mutants. Given that the amoeboid myosin Is possess both actin- and membrane-binding domains, the mutant phenotypes could be due to alterations in the generation and/or regulation of cell cortical tension. This has been directly tested by analyzing mutant Dictyostelium that either lacks or overexpresses various myosin Is, using micropipette aspiration techniques. Dictyostelium cells lacking only one myosin I have normal levels of cortical tension. However, myosin I double mutants have significantly reduced (50%) cortical tension, and those that mildly overexpress an amoeboid myosin I exhibit increased cortical tension. Treatment of either type of mutant with the lectin concanavalin A (ConA) that cross-links surface receptors results in significant increases in cortical tension, suggesting that the contractile activity of these myosin Is is not controlled by this stimulus. These results demonstrate that myosin Is work cooperatively to contribute substantially to the generation of resting cortical tension that is required for efficient cell migration and macropinocytosis.

A Dictyostelium myosin I plays a crucial role in regulating the frequency of pseudopods formed on the substratum

Analysis of the motile behavior of a strain of Dictyostelium lacking a myosin I, myoA, revealed that this mutant strain formed pseudopods and turned twice as frequently as wild type cells [Titus et al., 1993: Mol. Biol. Cell 4:233-246]. The basis for this aberrant behavior has been explored using three-dimensional reconstructions of translocating cells. Wild type cells form approximately 40% of pseudopods on the substratum and 60% off the substratum. The majority of pseudopods formed on the substratum initiate sharp turns while the majority of pseudopods formed off the substratum are retracted. Although myoA- cells form pseudopods at roughly twice the frequency of wild type cells, the increase in frequency is specific for only those pseudopods formed on the substratum. This increase is the basis for the aberrant increase in turning in myoA- cells. The selective increase in the frequency of pseudopods formed on the substratum correlates with a number of additional abnormalities in myoA- pseuodpod formation. First, myoA- cells can simultaneously extend more than one pseudopod, whereas wild type cells extend only one pseudopod at a time. Second, although wild type and myoA- pseudopods achieve the same final volumes, myoA- pseudopods grow at half the rate of wild type pseudopods and, therefore, take longer to achieve final volume. Third, while a wild type pseudopod grows in a continuous fashion, a myoA- pseudopod grows in a discontinuous fashion. Together, these results demonstrate that myoA plays a fundamental role in controlling the frequency of only those pseudopods formed on the substratum, and that maintenance of the normal frequency of pseudopod formation appears to be necessary for the normal velocity of cellular translocation, the normal frequency of turning, the normal rate of average pseudopod growth, and the high efficiency of chemotaxis. These results in turn indicate that pseudopod formation is precisely coordinated in space and time, and actin-associated proteins like myoA play key roles in coordination.

MYOSINS : MATCHING FUNCTIONS WITH MOTORS

It is an exciting time to be studying myosins and their roles in the function of cells and organisms. Past efforts aimed at finding new members of this family have now given way to a focus on identifying individual functions for each motor protein. These actin-based motors are now known to be intimately involved in the following processes: neurosensory function; vesicle trafficking; determinant partitioning; and cortical function. The following article reviews the inroads made into the functions of myosins in these processes over the past several years.

Unconventional myosins: new frontiers in actin-based motors.

The unconventional myosins are a superfamily of actin-based motors responsible for a rich array of intracellular motility events. Recent evidence suggests that these motors play important roles in cell migration, endocytosis and intracellular transport. Several genetic mutants have been identified whose abnormalities are the result of the loss of a specific myosin. This article describes how analysis of these mutants, coupled with basic studies of the intracellular localization and biochemical properties of individual myosins, is leading to a clearer understanding of the in vivo function of a number of these interesting motor proteins.

Motor proteins: Myosin V – the multi-purpose transport motor

Studies in yeast and mice suggest that myosin V participates in the directed transport of a number of distinct cargos to polarized regions of the cell; myosin V has also been implicated in the provision of materials for filopodial extension in neurons.

Intracellular motility: How can we all work together?

Recent results reinforce the view that actin-based and microtubule-based motility systems do not operate independently, but are used in coordinated fashion to determine intracellular localization of cargo such as organelles.

Dictyostelium discoideum myoJ: a member of a broadly defined myosin V class or a class XI unconventional myosin?

SummaryThe simple eukaryote Dictyostelium discoideum contains at least 12 unconventional myosin genes. Here we report the characterization of one of these, myoJ, a gene initially identified through a physical mapping screen. The myoJ gene encodes a high molecular weight myosin, and analysis of the available deduced amino acid sequence reveals that it possesses six IQ motifs and sequences typical of alpha helical coiled coils in the tail region. Therefore, myoJ is predicted to exist as a dimer with up to 12 associated light chains (six per heavy chain). The 7.8 kb myoJ mRNA is expressed all throughout the life cycle of D. discoideum. The myoJ gene has been disrupted and a phenotypic analysis of the mutant cells initiated. Finally, phylogenetic analysis of the head region reveals that myoJ is most similar to two plant myosin genes, Arabidopsis MYA1 and MYA2, that have been alternatively suggested to be either members of the myosin V class or founding members of the myosin XI class.

F‐actin Distribution of Dictyostelium Myosin I Double Mutants

The roles of the myosin I class of mechanoenzymes have been investigated by single and double gene knockout studies in the amoeba Dictyostelium discoideum. Cells lacking different myosin I pairs (myoA‐/myoB‐, myoB‐/myoC‐, and myoA‐/myoC‐) were examined with respect to their cytoskeletal organization. F‐actin localization by rhodamine‐phalloidin staining of cells indicates that the myoA‐/myoB‐, myoB‐/myoC‐, and myoA‐/myoC‐ cells appear to redistribute their F‐actin more slowly than wild type cells upon adhesion to a substrate. These studies suggest that Dictyostelium myoA, myoB, and myoC may have overlapping roles in maintaining the integrity or organization of the cortical membrane cytoskeleton.

A potential mechanism for regulating myosin I binding to membranes in vivo

Myosin Is are associated with specific membranes, however, the mechanism for regulating their intracellular localization is unclear. As a first step towards understanding this mechanism, membrane rebinding assays using Dictyostelium myoB were performed. Crude, cytosolic myoB bound to intact, but not to NaOH‐treated plasma membranes. In contrast, partially purified myoB binds to both intact and NaOH‐treated plasma membranes. Chemical cross‐linking of cytosolic myoB yielded several products, whereas none were found with the partially purified myoB. These results suggest a model where proteins regulating the specific binding of myoB to the plasma membrane may exist both in the cytosol and on the plasma membrane.

Science with a punch.

There are changes afoot in the way that US granting agencies, most notably the NIH, evaluate proposals. Change can be a good thing, and it is probably high time that the NIH reassessed how grants are evaluated. Frankly, I was becoming blase about the entire granting process. It seemed that once I became inured to the low probability of funding, competition from the mega-labs, and the shift towards preferring science that can be called ‘medically relevant’, all of the fun had been taken out of writing grants and reading the subsequent scathing evaluations. Now, the evaluation of science will include a more precise and specific ranking of several individual aspects of a proposal, one of which is the (real or imagined) significance or impact of the proposed research. Ah … life is worth living again!How can one argue with the notion that significant science should be funded and insignificant science should not? Although explicit ranking of the impact of the proposed research seems to be a great idea, surely reviewers have been overtly or covertly considering significance when rating grants all along. So why has this criterion now come out of the closet and how will it be applied evenly to all grant applications?One major reason for the change in the method of evaluation is that a mechanism for provoking reviewers into giving out a wider range of rankings seems to be needed, since so many grants score high and so few can be funded. If the evaluation can be broken down into smaller quanta there is a chance that the overall scores may have greater variability. But how can we put a number on such an inherently unpredictable aspect of science as its future significance? Yes, some projects have obvious impact, but one of the attractions of this business is the fact that there are times when biology is determined to undermine your nice, tidy, and oh-so-logical model in a new, fascinating and wholly unpredicted way. Apparently routine questions are forever coming up with unexpected answers. Will the new system reduce our ability to check and recheck the truth of our assumptions?Moving to the personal level, how can I assess the likely impact of my own research? How will I know that my work will affect the concepts or methods that drive my field, and how can I be sure that I am working on an important problem? I increasingly suspect that the fact that I think my science is interesting and important is irrelevant to the misguided people out there who don’t share my views. Is there a generally acceptable way to define high-impact science?One simple-minded criterion is to gauge significance using the publication record: the number of papers published, and where they are published. This criterion is convenient and readily quantifiable, with the added bonus that the editors and reviewers of the ‘correct’ journals do a good deal of the dirty work by deciding what areas of science are significant and which papers are likely to have an impact on the field. Perhaps in the future journals will be able to buy the right from the NIH to affix the label “The place where high-impact scientists publish!” to the front cover. But are we really happy with the idea that these faceless editors and reviewers who already have so much power will now have more? Are they really the best people to decide the overall scientific direction of the country? Perhaps.Its hard to guess which journal the paper is going to end up in before the work is even done, however. So can we define high-impact science by the area of research? Maybe the NIH should provide a list of high-impact topics, to alleviate confusion for those of us who might be unwittingly considering working in medium or low impact areas. In assembling this list, the NIH may want to consider whether high-impact science is work that even the man on the street has heard of, having an appeal not solely confined to the rarified circles of the scientist. By this criterion, research to improve the quality of the beers produced by the major brewers in the US would probably be the ultimate in high-impact biological science.However we decide to define high-impact science, there is one problem that nobody seems to have recognized yet. We need to provide therapy for people who are addicted to science of lesser significance. I envision a high-quality, extremely confidential clinic (the Watson & Crick Clinic for Significant Science) modeled along the lines of the Betty Ford treatment center for substance abusers. The anonymous patients would be offered a series of seminars alerting them to the warning signs of medium- or low-impact science, and would be taught to avoid self-destructive tendencies such as an affection for un-trendy research. This approach alone would greatly improve the perceived quality of science in many labs almost overnight.Of course it would be a good idea to shift the way that people develop projects in such a way as to make it more likely that truly significant work will be done. But will this change in NIH reviewing guidelines help or hinder this goal? I suspect the latter.