Myosin Va cooperates with PKA RIα to mediate maintenance of the endplate in vivo

Myosin V motor proteins facilitate recycling of synaptic receptors, including AMPA and acetylcholine receptors, in central and peripheral synapses, respectively. To shed light on the regulation of receptor recycling, we employed in vivo imaging of mouse neuromuscular synapses. We found that myosin Va cooperates with PKA on the postsynapse to maintain size and integrity of the synapse; this cooperation also regulated the lifetime of acetylcholine receptors. Myosin Va and PKA colocalized in subsynaptic enrichments. These accumulations were crucial for synaptic integrity and proper cAMP signaling, and were dependent on AKAP function, myosin Va, and an intact actin cytoskeleton. The neuropeptide and cAMP agonist, calcitonin-gene related peptide, rescued fragmentation of synapses upon denervation. We hypothesize that neuronal ligands trigger local activation of PKA, which in turn controls synaptic integrity and turnover of receptors. To this end, myosin Va mediates correct positioning of PKA in a postsynaptic microdomain, presumably by tethering PKA to the actin cytoskeleton.

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Myosin V attachment to cargo requires the tight association of two functional subdomains

The Journal of Cell Biology ◽

10.1083/jcb.200407146 ◽

2005 ◽

Vol 168 (3) ◽

pp. 359-364 ◽

Cited By ~ 30

Author(s):

Natasha Pashkova ◽

Natalie L. Catlett ◽

Jennifer L. Novak ◽

Guanming Wu ◽

Renne Lu ◽

...

Keyword(s):

Secretory Vesicle ◽

Intramolecular Interactions ◽

Tail Domain ◽

Myosin V ◽

Functional Complex ◽

Myosin Va ◽

Carboxyl Terminal ◽

Tight Association ◽

Simultaneous Expression

The myosin V carboxyl-terminal globular tail domain is essential for the attachment of myosin V to all known cargoes. Previously, the globular tail was viewed as a single, functional entity. Here, we show that the globular tail of the yeast myosin Va homologue, Myo2p, contains two structural subdomains that have distinct functions, namely, vacuole-specific and secretory vesicle–specific movement. Biochemical and genetic analyses demonstrate that subdomain I tightly associates with subdomain II, and that the interaction does not require additional proteins. Importantly, although neither subdomain alone is functional, simultaneous expression of the separate subdomains produces a functional complex in vivo. Our results suggest a model whereby intramolecular interactions between the globular tail subdomains help to coordinate the transport of multiple distinct cargoes by myosin V.

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Visualization of Melanosome Dynamics within Wild-Type and Dilute Melanocytes Suggests a Paradigm for Myosin V Function In Vivo

The Journal of Cell Biology ◽

10.1083/jcb.143.7.1899 ◽

1998 ◽

Vol 143 (7) ◽

pp. 1899-1918 ◽

Cited By ~ 295

Author(s):

Xufeng Wu ◽

Blair Bowers ◽

Kang Rao ◽

Qin Wei ◽

John A. Hammer

Keyword(s):

Wild Type Mouse ◽

Time Lapse ◽

Tail Domain ◽

Wild Type ◽

Myosin V ◽

Transport Models ◽

Myosin Va ◽

Cortical Shell ◽

Mutant Phenotypes

Unlike wild-type mouse melanocytes, where melanosomes are concentrated in dendrites and dendritic tips, melanosomes in dilute (myosin Va−) melanocytes are concentrated in the cell center. Here we sought to define the role that myosin Va plays in melanosome transport and distribution. Actin filaments that comprise a cortical shell running the length of the dendrite were found to exhibit a random orientation, suggesting that myosin Va could drive the outward spreading of melanosomes by catalyzing random walks. In contrast to this mechanism, time lapse video microscopy revealed that melanosomes undergo rapid (∼1.5 μm/s) microtubule-dependent movements to the periphery and back again. This bidirectional traffic occurs in both wild-type and dilute melanocytes, but it is more obvious in dilute melanocytes because the only melanosomes in their periphery are those undergoing this movement. While providing an efficient means to transport melanosomes to the periphery, this component does not by itself result in their net accumulation there. These observations, together with previous studies showing extensive colocalization of myosin Va and melanosomes in the actin-rich periphery, suggest a mechanism in which a myosin Va–dependent interaction of melanosomes with F-actin in the periphery prevents these organelles from returning on microtubules to the cell center, causing their distal accumulation. This “capture” model is supported by the demonstration that (a) expression of the myosin Va tail domain within wild-type cells creates a dilute-like phenotype via a process involving initial colocalization of tail domains with melanosomes in the periphery, followed by an ∼120-min, microtubule-based redistribution of melanosomes to the cell center; (b) microtubule-dependent melanosome movement appears to be damped by myosin Va; (c) intermittent, microtubule-independent, ∼0.14 μm/s melanosome movements are seen only in wild-type melanocytes; and (d) these movements do not drive obvious spreading of melanosomes over 90 min. We conclude that long-range, bidirectional, microtubule-dependent melanosome movements, coupled with actomyosin Va–dependent capture of melanosomes in the periphery, is the predominant mechanism responsible for the centrifugal transport and peripheral accumulation of melanosomes in mouse melanocytes. This mechanism represents an alternative to straightforward transport models when interpreting other myosin V mutant phenotypes.

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Drebrin Regulates Acetylcholine Receptor Clustering and Organization of Microtubules at the Postsynaptic Machinery

International Journal of Molecular Sciences ◽

10.3390/ijms22179387 ◽

2021 ◽

Vol 22 (17) ◽

pp. 9387

Author(s):

Paloma Alvarez-Suarez ◽

Natalia Nowak ◽

Anna Protasiuk-Filipunas ◽

Hiroyuki Yamazaki ◽

Tomasz J. Prószyński ◽

...

Keyword(s):

Actin Cytoskeleton ◽

Acetylcholine Receptors ◽

Neuromuscular Junctions ◽

Microtubule Network ◽

Postsynaptic Protein ◽

Cultured Myotubes ◽

The Brain

Proper muscle function depends on the neuromuscular junctions (NMJs), which mature postnatally to complex “pretzel-like” structures, allowing for effective synaptic transmission. Postsynaptic acetylcholine receptors (AChRs) at NMJs are anchored in the actin cytoskeleton and clustered by the scaffold protein rapsyn, recruiting various actin-organizing proteins. Mechanisms driving the maturation of the postsynaptic machinery and regulating rapsyn interactions with the cytoskeleton are still poorly understood. Drebrin is an actin and microtubule cross-linker essential for the functioning of the synapses in the brain, but its role at NMJs remains elusive. We used immunohistochemistry, RNA interference, drebrin inhibitor 3,5-bis-trifluoromethyl pyrazole (BTP2) and co-immunopreciptation to explore the role of this protein at the postsynaptic machinery. We identify drebrin as a postsynaptic protein colocalizing with the AChRs both in vitro and in vivo. We also show that drebrin is enriched at synaptic podosomes. Downregulation of drebrin or blocking its interaction with actin in cultured myotubes impairs the organization of AChR clusters and the cluster-associated microtubule network. Finally, we demonstrate that drebrin interacts with rapsyn and a drebrin interactor, plus-end-tracking protein EB3. Our results reveal an interplay between drebrin and cluster-stabilizing machinery involving rapsyn, actin cytoskeleton, and microtubules.

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Induction of insulin resistance in vivo by amylin and calcitonin gene-related peptide

Diabetes ◽

10.2337/diabetes.39.2.260 ◽

1990 ◽

Vol 39 (2) ◽

pp. 260-265 ◽

Cited By ~ 46

Author(s):

J. M. Molina ◽

G. J. Cooper ◽

B. Leighton ◽

J. M. Olefsky

Keyword(s):

Insulin Resistance ◽

Calcitonin Gene Related Peptide ◽

Related Peptide ◽

Calcitonin Gene

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Differential pial and penetrating arterial responses examined by optogenetic activation of astrocytes and neurons

Journal of Cerebral Blood Flow & Metabolism ◽

10.1177/0271678x211010355 ◽

2021 ◽

pp. 0271678X2110103

Author(s):

Nao Hatakeyama ◽

Miyuki Unekawa ◽

Juri Murata ◽

Yutaka Tomita ◽

Norihiro Suzuki ◽

...

Keyword(s):

Cortical Neurons ◽

Acetylcholine Receptors ◽

Step Function ◽

Muscarinic Acetylcholine Receptors ◽

Cell Type ◽

Function Type ◽

Neuron Activation ◽

Cell Type Specific ◽

Arterial Responses

A variety of brain cells participates in neurovascular coupling by transmitting and modulating vasoactive signals. The present study aimed to probe cell type-dependent cerebrovascular (i.e., pial and penetrating arterial) responses with optogenetics in the cortex of anesthetized mice. Two lines of the transgenic mice expressing a step function type of light-gated cation channel (channelrhodopsine-2; ChR2) in either cortical neurons (muscarinic acetylcholine receptors) or astrocytes (Mlc1-positive) were used in the experiments. Photo-activation of ChR2-expressing astrocytes resulted in a widespread increase in cerebral blood flow (CBF), extending to the nonstimulated periphery. In contrast, photo-activation of ChR2-expressing neurons led to a relatively localized increase in CBF. The differences in the spatial extent of the CBF responses are potentially explained by differences in the involvement of the vascular compartments. In vivo imaging of the cerebrovascular responses revealed that ChR2-expressing astrocyte activation led to the dilation of both pial and penetrating arteries, whereas ChR2-expressing neuron activation predominantly caused dilation of the penetrating arterioles. Pharmacological studies showed that cell type-specific signaling mechanisms participate in the optogenetically induced cerebrovascular responses. In conclusion, pial and penetrating arterial vasodilation were differentially evoked by ChR2-expressing astrocytes and neurons.

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Cofactor bypass variants reveal a conformational control mechanism governing cell wall polymerase activity

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1524538113 ◽

2016 ◽

Vol 113 (17) ◽

pp. 4788-4793 ◽

Cited By ~ 21

Author(s):

Monica Markovski ◽

Jessica L. Bohrhunter ◽

Tania J. Lupoli ◽

Tsuyoshi Uehara ◽

Suzanne Walker ◽

...

Keyword(s):

Polymerase Activity ◽

Activation Mechanism ◽

Phenotypic Analysis ◽

Outer Membranes ◽

Division Site ◽

Physiological Relevance ◽

Membrane Lipoproteins ◽

Shed Light

To fortify their cytoplasmic membrane and protect it from osmotic rupture, most bacteria surround themselves with a peptidoglycan (PG) exoskeleton synthesized by the penicillin-binding proteins (PBPs). As their name implies, these proteins are the targets of penicillin and related antibiotics. We and others have shown that the PG synthases PBP1b and PBP1a ofEscherichia colirequire the outer membrane lipoproteins LpoA and LpoB, respectively, for their in vivo function. Although it has been demonstrated that LpoB activates the PG polymerization activity of PBP1b in vitro, the mechanism of activation and its physiological relevance have remained unclear. We therefore selected for variants of PBP1b (PBP1b*) that bypass the LpoB requirement for in vivo function, reasoning that they would shed light on LpoB function and its activation mechanism. Several of these PBP1b variants were isolated and displayed elevated polymerization activity in vitro, indicating that the activation of glycan polymer growth is indeed one of the relevant functions of LpoB in vivo. Moreover, the location of amino acid substitutions causing the bypass phenotype on the PBP1b structure support a model in which polymerization activation proceeds via the induction of a conformational change in PBP1b initiated by LpoB binding to its UB2H domain, followed by its transmission to the glycosyl transferase active site. Finally, phenotypic analysis of strains carrying a PBP1b* variant revealed that the PBP1b–LpoB complex is most likely not providing an important physical link between the inner and outer membranes at the division site, as has been previously proposed.

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N-cholinergic facilitation of glutamate release from an individual retinotectal fiber in frog

Visual Neuroscience ◽

10.1017/s0952523801184051 ◽

2001 ◽

Vol 18 (4) ◽

pp. 549-558 ◽

Cited By ~ 16

Author(s):

A. KURAS ◽

N. GUTMANIENĖ

Keyword(s):

Nicotinic Acetylcholine Receptors ◽

Action Potentials ◽

Acetylcholine Receptors ◽

Kynurenic Acid ◽

Glutamate Release ◽

Synaptic Potentials ◽

Lower Vertebrates ◽

Optic Fibers ◽

Retinotectal Transmission

Nicotinic acetylcholine receptors are localized on retinotectal axons' terminals in lower vertebrates. The effects of activation of these receptors by endogenous acetylcholine were observed under stimulation of mass optic fibers. This study was designed to determine whether endogenous acetylcholine facilitates frog retinotectal transmission, provided only the synapses of an individual optic axon are activated, and to evaluate the feasible extent of nicotinic facilitation in these synapses by applied agonist. To this end, the effects of cholinergic drugs on the extracellular action and synaptic potentials recorded from the terminal arborization of a separate retinotectal fiber (in layer F of the tectum) were investigated in vivo. Glutamatergic nature of retinotectal synapses was reexamined by treatment with kynurenic acid. Both kynurenic acid (0.25–1 mM) and d-tubocurarine chloride (10–15 μM) significantly depressed the synaptic potentials. Carbamylcholine chloride (50–150 μM) evoked a large augmentation of the synaptic potentials and a slight but statistically significant decrease of the action potentials. D-tubocurarine reduced the effect of carbamylcholine. Pilocarpine hydrochloride (50 μM) had only a weak effect. The paired-pulse facilitation of the synaptic potentials changed significantly under the action of carbamylcholine and d-tubocurarine. The obtained results suggest that the glutamate release from activated synapses of individual retinotectal axons is facilitated by endogenous acetylcholine via presynaptic nicotinic receptors. Under used stimulation conditions, this modulation mechanism was employed only partially since its activation by applied carbamylcholine could enhance synaptic transmission up to 2.8 times.

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Globular and asymmetric acetylcholinesterase in frog muscle basal lamina sheaths.

The Journal of Cell Biology ◽

10.1083/jcb.102.3.762 ◽

1986 ◽

Vol 102 (3) ◽

pp. 762-768 ◽

Cited By ~ 12

Author(s):

M Nicolet ◽

M Pinçon-Raymond ◽

F Rieger

Keyword(s):

Basal Lamina ◽

Acetylcholine Receptors ◽

Muscle Fibers ◽

Frog Muscle ◽

Motor Endplate ◽

Molecular Forms ◽

Nonionic Detergent ◽

Plasmic Membrane ◽

Triton X

After denervation in vivo, the frog cutaneus pectoris muscle can be led to degenerate by sectioning the muscle fibers on both sides of the region rich in motor endplate, leaving, 2 wk later, a muscle bridge containing the basal lamina (BL) sheaths of the muscle fibers (28). This preparation still contains various tissue remnants and some acetylcholine receptor-containing membranes. A further mild extraction by Triton X-100, a nonionic detergent, gives a pure BL sheath preparation, devoid of acetylcholine receptors. At the electron microscope level, this latter preparation is essentially composed of the muscle BL with no attached plasmic membrane and cellular component originating from Schwann cells or macrophages. Acetylcholinesterase is still present in high amounts in this BL sheath preparation. In both preparations, five major molecular forms (18, 14, 11, 6, and 3.5 S) can be identified that have either an asymmetric or a globular character. Their relative amount is found to be very similar in the BL and in the motor endplate-rich region of control muscle. Thus, observations show that all acetylcholinesterase forms can be accumulated in frog muscle BL.

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In Vivo 17β-Estradiol Treatment Contributes to Podocyte Actin Stabilization in Female db/db Mice

Endocrinology ◽

10.1210/en.2012-1637 ◽

2012 ◽

Vol 153 (12) ◽

pp. 5888-5895 ◽

Cited By ~ 8

Author(s):

Paola Catanuto ◽

Alessia Fornoni ◽

Simone Pereira-Simon ◽

Fayi Wu ◽

Kerry L. Burnstein ◽

...

Keyword(s):

Actin Cytoskeleton ◽

Transcriptional Activation ◽

Cell Types ◽

Proper Function ◽

Estradiol Treatment ◽

Diabetic Glomerulosclerosis ◽

Podocyte Damage ◽

Rac1 Activity ◽

17Β Estradiol

Abstract We recently showed that 17β-estradiol (E2) treatment ameliorated type 2 diabetic glomerulosclerosis in mice in part by protecting podocyte structure and function. Progressive podocyte damage is characterized by foot process effacement, vacuolization, detachment of podocytes from the glomerular basement membrane, and apoptosis. In addition, podocytes are highly dependent on the preservation of their actin cytoskeleton to ensure proper function and survival. Because E2 administration prevented podocyte damage in our study on diabetic db/db mice and has been shown to regulate both actin cytoskeleton and apoptosis in other cell types and tissues, we investigated whether actin remodeling and apoptosis were prevented in podocytes isolated from E2-treated diabetic db/db mice. We performed G-actin/F-actin assays, Western analysis for Hsp25 expression, Ras-related C3 botulinum toxin substrate 1 (Rac1) activity, and apoptosis assays on previously characterized podocytes isolated from both in vivo-treated placebo and E2 female db/db mice. We found that in vivo E2 protects against a phenotype change in the cultured podocytes characterized by a percent increase of F-actin vs. G-actin, suppression of Hsp25 expression and transcriptional activation, increase of Rac1 activity, and decreased apoptotic intermediates. We conclude from these studies that E2 treatment protects against podocyte damage and may prevent/reduce diabetes-induced kidney disease.

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