Laura K. Gunther

Coupling of two non-processive myosin 5c dimers enables processive stepping along actin filaments

Myosin 5c (Myo5c) is a low duty ratio, non-processive motor unable to move continuously along actin filaments though it is believed to participate in secretory vesicle trafficking in vertebrate cells. Here, we measured the ATPase kinetics of Myo5c dimers and tested the possibility that the coupling of two Myo5c molecules enables processive movement. Steady-state ATPase activity and ADP dissociation kinetics demonstrated that a dimer of Myo5c-HMM (double-headed heavy meromyosin 5c) has a 6-fold lower Km for actin filaments than Myo5c-S1 (single-headed myosin 5c subfragment-1), indicating that the two heads of Myo5c-HMM increase F-actin-binding affinity. Nanometer-precision tracking analyses showed that two Myo5c-HMM dimers linked with each other via a DNA scaffold and moved processively along actin filaments. Moreover, the distance between the Myo5c molecules on the DNA scaffold is an important factor for the processive movement. Individual Myo5c molecules in two-dimer complexes move stochastically in 30–36 nm steps. These results demonstrate that two dimers of Myo5c molecules on a DNA scaffold increased the probability of rebinding to F-actin and enabled processive steps along actin filaments, which could be used for collective cargo transport in cells.

Actin Structure-Dependent Stepping of Myosin 5a and 10 during Processive Movement

How myosin 10, an unconventional myosin, walks processively along actin is still controversial. Here, we used single molecule fluorescence techniques, TIRF and FIONA, to study the motility and the stepping mechanism of dimerized myosin 10 heavy-meromyosin-like fragment on both single actin filaments and two-dimensional F-actin rafts cross-linked by fascin or α-actinin. As a control, we also tracked and analyzed the stepping behavior of the well characterized processive motor myosin 5a. We have shown that myosin 10 moves processively along both single actin filaments and F-actin rafts with a step size of 31 nm. Moreover, myosin 10 moves more processively on fascin-F-actin rafts than on α-actinin-F-actin rafts, whereas myosin 5a shows no such selectivity. Finally, on fascin-F-actin rafts, myosin 10 has more frequent side steps to adjacent actin filaments than myosin 5a in the F-actin rafts. Together, these results reveal further single molecule features of myosin 10 on various actin structures, which may help to understand its cellular functions.

The actin-bundling protein TRIOBP-4 and -5 promotes the motility of pancreatic cancer cells

TRIOBP isoforms 4 and 5 (TRIOBP-4/-5) are an actin-bundling protein associated with hearing loss. Here, we showed that TRIOBP-4/-5 was up-regulated in human pancreatic carcinoma cells. Knockdown of TRIOBP-4/-5 led to a loss of filopodia and a decrease in cell motility. Confocal microscopy showed that re-expression of GFP-TRIOBP-4 or -5 restored the filopodial formation in TRIOBP-4/-5-deficient PANC-1 cells. Finally, TRIOBP-4/-5 was shown to be overexpressed in human pancreatic cancer tissues. These results demonstrate a novel role of TRIOBP-4/-5 that promotes the motility of pancreatic cancer cells via regulating actin cytoskeleton reorganization in the filopodia of the cells.

R1 motif is the major actin-binding domain of TRIOBP-4.

TRIOBP is an actin-bundling protein. Mutations of TRIOBP are associated with human deafness DFNB28. In vitro, TRIOBP isoform 4 (TRIOBP-4) forms dense F-actin bundles resembling the inner ear hair cell rootlet structure. Deletion of TRIOBP isoforms 4 and 5 leads to hearing loss in mice due to the absence of stereocilia rootlets. The mechanism of actin bundle formation by TRIOBP is not fully understood. The amino acid sequences of TRIOBP isoforms 4 and 5 contain two repeated motifs, referred to here as R1 and R2. To examine the potential role of R1 and R2 motifs in F-actin binding, we generated TRIOBP-4 mutant proteins deleted for R1 and/or R2, and then assessed their actin-binding activity and bundle formation in vitro using actin cosedimentation assays, and fluorescence and electron microscopy. Cellular distributions of the TRIOBP-4 mutants were examined by confocal microscopy. We showed that deletion of both R1 and R2 motifs completely disrupted the actin binding/bundling activities of TRIOBP-4 and impaired its localization to cellular actin cytoskeleton structures. By contrast, TRIOBP-4, lacking only R2 motif, retained its F-actin bundling ability and remained localized to actin filaments in cells, similar to full length TRIOBP-4. On the contrary, the R1 motif-deleted TRIOBP-4 mutant, which mainly consists of the R2 motif, formed thin F-actin bundles in vitro but failed to colocalize to actin filaments in cells. These results indicate that R1 motif is the major actin-binding domain of TRIOBP-4, and the binding of R2 motif with actin filaments is nonspecific.

Effect of N-Terminal Extension of Cardiac Troponin I on the Ca2+ Regulation of ATP Binding and ADP Dissociation of Myosin II in Native Cardiac Myofibrils

Cardiac troponin I (cTnI) has a unique N-terminal extension that plays a role in modifying the calcium regulation of cardiac muscle contraction. Restrictive cleavage of the N-terminal extension of cTnI occurs under stress conditions as a physiological adaptation. Recent studies have shown that in comparison with controls, transgenic mouse cardiac myofibrils containing cTnI lacking the N-terminal extension (cTnI-ND) had a lower sensitivity to calcium activation of ATPase, resulting in enhanced ventricular relaxation and cardiac function. To investigate which step(s) of the ATPase cycle is regulated by the N-terminal extension of cTnI, here we studied the calcium dependence of cardiac myosin II ATPase kinetics in isolated cardiac myofibrils. ATP binding and ADP dissociation rates were measured by using stopped-flow spectrofluorimetry with mant-dATP and mant-dADP, respectively. We found that the second-order mant-dATP binding rate of cTnI-ND mouse cardiac myofibrils was 3-fold faster than that of wild-type myofibrils at low Ca(2+) concentrations. The ADP dissociation rate of cTnI-ND myofibrils was positively dependent on calcium concentration, while the wild-type controls were not significantly affected. These data from experiments using native cardiac myofibrils under physiological conditions indicate that modification of the N-terminal extension of cTnI plays a role in the calcium regulation of the kinetics of actomyosin ATPase.

Large Vesicle Moves in Rolling Manner by Myosin 5C along Actin Tracks

Class 5 myosins are actin-based molecular motors implicated in intracellular cargo transport. In vertebrates, myosin 5 (Myo5) consists of three isoforms, named myosin 5a, 5b, and 5c. The ability of myosin V molecules to move continuously along actin filaments (i.e. processivity) is required for efficient cargo transport in cells. This physical property has been well addressed in Myo5a and 5b, which demonstrate processive movement as a single molecule. In contrast, Myo5c shows no processivity as a single molecule, although it is found in cargo and is believed to participate in cargo transports. This raises the possibility that multiple Myo5c molecules should transport cargos. Recent studies have shown that Myo5c binds to Zymogen granule (ZGs) in pancreatic cells and transports ZGs to cell periphery. Purified ZGs was investigated the movement manner in vitro. The velocity and run-length of ZGs along actin track was measured (99 nm/sec and 1255 nm, respectively). We identified that the movement manner of ZGs move in rolling manner instead of gliding along actin tracks. Mathematical model of multiple Myo5c motors ensemble applied to understand the movement behavior vs. number of Myo5c on the vesicle.