miR-409-3p represses Cited2 to refine neocortical layer V projection neuron identity.

The evolutionary emergence of the corticospinal tract and corpus callosum are thought to underpin the expansion of complex motor and cognitive abilities in mammals. Molecular mechanisms regulating development of the neurons whose axons comprise these tracts, the corticospinal and callosal projection neurons, remain incompletely understood. Our previous work identified a genomic cluster of microRNAs (miRNAs), Mirg/12qF1, that is unique to placental mammals and specifically expressed by corticospinal neurons, and excluded from callosal projection neurons, during development. We found that one of these, miR-409-3p, can convert layer V callosal into corticospinal projection neurons, acting in part through repression of the transcriptional regulator Lmo4. Here we show that miR-409-3p also directly represses the transcriptional co-regulator Cited2, which is highly expressed by callosal projection neurons from the earliest stages of neurogenesis. Cited2 is highly expressed by intermediate progenitor cells (IPCs) in the embryonic neocortex while Mirg, which encodes miR-409-3p, is excluded from these progenitors. miR-409-3p gain-of-function (GOF) in IPCs results in a phenocopy of established Cited2 loss-of-function (LOF). At later developmental stages, both miR-409-3p GOF and Cited2 LOF promote the expression of corticospinal at the expense of callosal projection neuron markers in layer V. Taken together, this work identifies previously undescribed roles for miR-409-3p in controlling IPC numbers and for Cited2 in controlling callosal fate. Thus, miR-409-3p, possibly in cooperation with other Mirg/12qF1 miRNAs, represses Cited2 as part of the multifaceted regulation of the refinement of neuronal cell fate within layer V, combining molecular regulation at multiple levels in both progenitors and post-mitotic neurons.

A binding protein regulates myosin-7a dimerization and actin bundle assembly.

Myosin-7a, despite being monomeric in isolation, plays roles in organizing actin-based cell protrusions such as filopodia, microvilli and stereocilia, as well as transporting cargoes within them. Here, we identify a binding protein for Drosophila myosin-7a termed M7BP, and describe how M7BP assembles myosin-7a into a motile complex that enables cargo translocation and actin cytoskeletal remodeling. M7BP binds to the autoinhibitory tail of myosin-7a, extending the molecule and activating its ATPase activity. Single-molecule reconstitution show that M7BP enables robust motility by complexing with myosin-7a as 2:2 translocation dimers in an actin-regulated manner. Meanwhile, M7BP tethers actin, enhancing complex's processivity and driving actin-filament alignment during processive runs. Finally, we show that myosin-7a-M7BP complex assembles actin bundles and filopodia-like protrusions while migrating along them in living cells. Together, these findings provide insights into the mechanisms by which myosin-7a functions in actin protrusions.

An evolutionarily acquired microRNA shapes development of mammalian cortical projections.

The corticospinal tract is unique to mammals and the corpus callosum is unique to placental mammals (eutherians). The emergence of these structures is thought to underpin the evolutionary acquisition of complex motor and cognitive skills. Corticospinal motor neurons (CSMN) and callosal projection neurons (CPN) are the archetypal projection neurons of the corticospinal tract and corpus callosum, respectively. Although a number of conserved transcriptional regulators of CSMN and CPN development have been identified in vertebrates, none are unique to mammals and most are coexpressed across multiple projection neuron subtypes. Here, we discover 17 CSMN-enriched microRNAs (miRNAs), 15 of which map to a single genomic cluster that is exclusive to eutherians. One of these, miR-409-3p, promotes CSMN subtype identity in part via repression of LMO4, a key transcriptional regulator of CPN development. In vivo, miR-409-3p is sufficient to convert deep-layer CPN into CSMN. This is a demonstration of an evolutionarily acquired miRNA in eutherians that refines cortical projection neuron subtype development. Our findings implicate miRNAs in the eutherians' increase in neuronal subtype and projection diversity, the anatomic underpinnings of their complex behavior.

Regulation of myosin 5a and myosin 7a.

The myosin superfamily is diverse in its structure, kinetic mechanisms and cellular function. The enzymatic activities of most myosins are regulated by some means such as Ca2+ ion binding, phosphorylation or binding of other proteins. In the present review, we discuss the structural basis for the regulation of mammalian myosin 5a and Drosophila myosin 7a. We show that, although both myosins have a folded inactive state in which domains in the myosin tail interact with the motor domain, the details of the regulation of these two myosins differ greatly.

Time course and strain dependence of ADP release during contraction of permeabilized skeletal muscle fibers.

A phosphorylated, single cysteine mutant of nucleoside diphosphate kinase, labeled with N-[2-(iodoacetamido)ethyl]-7-diethylaminocoumarin-3-carboxamide (P approximately NDPK-IDCC), was used as a fluorescence probe for time-resolved measurement of changes in [MgADP] during contraction of single permeabilized rabbit psoas fibers. The dephosphorylation of the phosphorylated protein by MgADP occurs within the lattice environment of permeabilized fibers with a second-order rate constant at 12 degrees C of 10(5) M(-1) s(-1). This dephosphorylation is accompanied by a change in coumarin fluorescence. We report the time course of P approximately NDPK-IDCC dephosphorylation during the period of active isometric force redevelopment after quick release of fiber strain at pCa(2+) of 4.5. After a rapid length decrease of 0.5% was applied to the fiber, the extra NDPK-IDCC produced during force recovery, above the value during the approximately steady state of isometric contraction, was 2.7 +/- 0.6 microM and 4.7 +/- 1.5 microM at 12 and 20 degrees C, respectively. The rates of P approximately NDPK-IDCC dephosphorylation during force recovery were 28 and 50 s(-1) at 12 and 20 degrees C, respectively. The time courses of isometric force and P approximately NDPK-IDCC dephosphorylation were simulated using a seven-state reaction scheme. Relative isometric force was modeled by changes in the occupancy of strongly bound A.M.ADP.P(i) and A.M.ADP states. A strain-sensitive A.M.ADP isomerization step was rate-limiting (3-6 s(-1)) in the cross-bridge turnover during isometric contraction. At 12 degrees C, the A.M.ADP.P(i) and the pre- and postisomerization A.M.ADP states comprised 56%, 38%, and 7% of the isometric force-bearing AM states, respectively. At 20 degrees C, the force-bearing A.M.ADP.P(i) state was a lower proportion of the total force-bearing states (37%), whereas the proportion of postisomerization A.M.ADP states was higher (19%). The simulations suggested that release of cross-bridge strain caused rapid depopulation of the preisomerization A.M.ADP state and transient accumulation of MgADP in the postisomerization A.M.ADP state. Hence, the strain-sensitive isomerization of A.M.ADP seems to explain the rate of change of P approximately NDPK-IDCC dephosphorylation during force recovery. The temperature-dependent isometric distribution of myosin states is consistent with the previous observation of a small decrease in amplitude of the P(i) transient during force recovery at 20 degrees C and the current observation of an increase in amplitude of the ADP-sensitive NDPK-IDCC transient.

A FERM domain autoregulates Drosophila myosin 7a activity.

Full-length Drosophila myosin 7a (myosin 7a-FL) has a complex tail containing a short predicted coiled coil followed by a MyTH4-FERM domain, an SH3 domain, and a C-terminal MyTH4-FERM domain. Myosin 7a-FL expressed in Sf9 cells is monomeric despite the predicted coiled coil. We showed previously that Subfragment-1 (S1) from this myosin has MgATPase of V(max) approximately 1 s(-1) and K(ATPase) approximately 1 microM actin. We find that myosin 7a-FL has V(max) similar to S1 but K(ATPase) approximately 30 microM. Thus, at low actin concentrations (5 microM), the MgATPase of S1 is fully activated, whereas that of myosin 7a-FL is low, suggesting that the tail regulates activity. Electron microscopy of myosin 7a-FL with ATP shows the tail is tightly bent back against the motor domain. Myosin 7a-FL extends at either high ionic strength or without ATP, revealing the motor domain, lever, and tail. A series of C-terminal truncations show that deletion of 99 aa (the MyTH7 subdomain of the C-terminal FERM domain) is sufficient to abolish bending, and the K(ATPase) is then similar to S1. This region is highly conserved in myosin 7a. We found that a double mutation in it, R2140A-K2143A, abolishes bending and reduces K(ATPase) to S1 levels. In addition, the expressed C-terminal FERM domain binds actin with K(d) approximately 30 microM regardless of ATP, similar to the K(ATPase) value for myosin 7a-FL. We propose that at low cellular actin concentrations, myosin 7a-FL is bent and inactive, but at high actin concentrations, it is unfolded and active because the C-terminal FERM domain binds to actin.

The "roll and lock" mechanism of force generation in muscle.

Muscle force results from the interaction of the globular heads of myosin-II with actin filaments. We studied the structure-function relationship in the myosin motor in contracting muscle fibers by using temperature jumps or length steps combined with time-resolved, low-angle X-ray diffraction. Both perturbations induced simultaneous changes in the active muscle force and in the extent of labeling of the actin helix by stereo-specifically bound myosin heads at a constant total number of attached heads. The generally accepted hypothesis assumes that muscle force is generated solely by tilting of the lever arm, or the light chain domain of the myosin head, about its catalytic domain firmly bound to actin. Data obtained suggest an additional force-generating step: the "roll and lock" transition of catalytic domains of non-stereo-specifically attached heads to a stereo-specifically bound state. A model based on this scheme is described to quantitatively explain the data.