Phorbol ester induces actin cytoskeleton rearrangements in cultured vascular smooth muscle cells. Calponin and SM22 alpha are major components of differentiated smooth muscle and potential regulators of actin cytoskeleton interactions. Here we show that actin fibers decorated with h1 CaP remain stable, whereas SM22 alpha-decorated actin bundles undergo rapid reorganization into podosomes within 30 min of PDBu exposure. Ectopic expression of GFP alpha-actinin had no effect on the stability of the actin cytoskeleton and alpha-actinin was transported rapidly into PDBu-induced podosomes. Our results demonstrate the involvement of CaP and SM22 alpha in coordinating the balance between stabilization and dynamics of the actin cytoskeleton in mammalian smooth muscle. We provide evidence for the existence of two functionally distinct actin filament populations and introduce a molecular mechanism for the stabilization of the actin cytoskeleton by the unique actin-binding interface formed by calponin family-specific CLIK23 repeats.

Calponin repeats regulate actin filament stability and formation of podosomes in smooth muscle cells.

Conventional protein kinase C mediates phorbol-dibutyrate-induced cytoskeletal remodeling in a7r5 smooth muscle cells.

Protein Kinase C δ (δPKC)-Annexin V Interaction A REQUIRED STEP IN δPKC TRANSLOCATION AND FUNCTION

An increase in the cytosolic Ca2+ concentration is a prerequisite in activation of contractile activity of smooth muscle. The shape of the Ca2+-signal is determined by spatial distribution and kinetics of Ca2+-binding sites in the cell. The increase in cytosolic Ca2+ activates myosin light chain kinase (MLCK) which in turn phosphorylates the regulatory light chains of myosin II. This Ca2+-dependent MLC20 phosphorylation is modulated in a Ca2+-independent manner by inhibiting the constitutive active myosin light chain phosphatase mediated by the monomeric GTPase Rho and the Rho-associated kinase as well as protein kinase C or by increasing its activity through cGMP. Furthermore, the activity of MLCK may be decreased due to phosphorylation by CaM kinase II and perhaps p21 activated protein kinase. Hence, smooth muscle tone appears to be regulated by a network of activating and inactivating intracellular signaling cascades which not only show a temporal but also a spatial activation pattern.

Ca2+-dependent and Ca2+-independent regulation of smooth muscle contraction.

Aberrant cardiomyocyte microtubule growth is a feature of pressure overload induced cardiac hypertrophy believed to contribute to left ventricular (LV) dysfunction Microtubule Actin Cross-linking Factor 1 (MACF1/Acf7) is a 600 kd spectraplakin that stabilizes and guides microtubule growth along actin filaments MACF1 is expressed in the heart, but its impact on cardiac microtubules, and how this influences cardiac structure, function, and adaptation to hemodynamic overload is unknown Here we used inducible cardiac-specific MACF1 knockout mice (MACF1 KO) to determine the impact of MACF1 on cardiac microtubules and adaptation to pressure overload (transverse aortic constriction (TAC)In adult mouse hearts, MACF1 expression was low under basal conditions, but increased significantly in response to TAC While MACF1 KO had no observable effect on heart size or function under basal conditions, MACF1 KO exacerbated TAC induced LV hypertrophy, LV dilation and contractile dysfunction Interestingly, subcellular fractionation of ventricular lysates revealed that MACF1 KO altered microtubule distribution in response to TAC, so that more tubulin was associated with the cell membrane fraction Moreover, TAC induced microtubule redistribution into this cell membrane fraction in both WT and MACF1 KO mice correlated strikingly with the level of contractile dysfunction (r2 = 0786, p<001) MACF1 disruption also resulted in reduction of membrane caveolin 3 levels, and increased levels of membrane PKCα and β1 integrin after TAC, suggesting MACF1 function is important for spatial regulation of several physiologically relevant signaling proteins during hypertrophy Together, these data identify for the first time, a role for MACF1 in cardiomyocyte microtubule distribution and in adaptation to hemodynamic overload

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Microtubule Actin Cross-linking Factor 1 regulates cardiomyocyte microtubule distribution and adaptation to hemodynamic overload.

Reports from numerous laboratories suggest that protein kinase C (PKC) translocation to substrate target sites may vary depending on cell type and experimental conditions. We have proposed that acutely variable targeting of PKC to different substrate sites could greatly expand the functional properties of individual isoforms in individual cell types (Li et al., 2001). Confocal microscopy and PKC alpha-enhanced green fluorescent protein (PKC alpha-EGFP) fusion protein expression were utilized to investigate the spatial and temporal pattern of PKC alpha translocation to different stimulating agents in A7r5 smooth muscle cells. Phorbol 12, 13 dibutyrate (PDBu 10(-8) M) caused a slow but irreversible relocation of the fusion protein from the cytosol to the plasmalemma. By comparison, thapsigargin (10(-5) M) and A23 187 (2 x 10(-5) M) induced a rapidly transient translocation to the cell membrane which was completed within 4 min. In contrast to these agents, angiotensin II (Ang II, 10(-6) M) caused only partial relocalization of cytosolic PKC alpha-EGFP to brightly fluorescing patches at the cell periphery. Localization at peripheral patches was completed within seconds and the fusion protein returned to the cytosol within 2 min. The PKC inhibitor staurosporine blocked cellular contraction to PDBu but not A(23 187) and had no effect on PKC alpha-EGFP translocation. By comparison, the calcium chelators EDTA and BAPTA-AM blocked the contraction to A(23 187), attenuated the contraction to PDBu, and abolished the translocation of PKC alpha-EGFP by both agents. The results show that in a single cell type the spatial and temporal characteristics of individual PKC isoform translocation may differ markedly. This further suggests the existence of potentially complex mechanisms which regulate the rate and location of target site availability.

PKC‐α shows variable patterns of translocation in response to different stimulatory agents

The translocation of protein kinase C (PKC) isozymes from their inactive cell locus to a variety of cytoskeletal, organelle, and plasmalemmal sites is thought to play an important role in their activation and substrate specificity. We have utilized confocal microscopy to compare phorbol 12, 13 dibutyrate (PDB) - stimulated translocation of PKCalpha in cultured cells derived from rat vascular smooth muscle. In enzymatically dispersed, passaged smooth muscle cells, PKCalpha was uniformly distributed throughout the unstimulated cell. PDB stimulation resulted in extensive association of the PKCalpha into filamentous strands with subsequent accumulation of the isoform in the peri-nuclear region of the cell. Dual immunostaining indicated that PKCalpha was extensively colocalized with microtubules in the interval immediately following PDB stimulation but was largely disassociated from microtubules at 10 min, at which time the translocation of PKCalpha to the peri-nucleus/nucleus was nearly complete. It was further found that the use of colchicine to disrupt the microtubules caused the loss of PKCalpha translocation to the peri-nuclear region. By comparison, cytochalasin B disruption of actin microfilaments had no significant effect on this parameter. The data suggest that PDB stimulation results in a transient association of PKCalpha with cell microtubules and that the microtubules play an important role in the translocation of PKCalpha from the cytosol in passaged cells derived from rat aortic smooth muscle.

PKCα translocation is microtubule‐dependent in passaged smooth muscle cells

The subcellular translocation of PKCα was studied in A7r5 cells by confocal microscopy through use of standard immunohistologic staining and PKCα-enhanced green fluorescent protein (PKCα–EGFP) fusion protein expression. The results from both methods were consistent in indicating that PKCα, observed to be diffusely distributed in the unstimulated cell, was translocated primarily to either the perinuclear region of the cell or to subplasmalemmal sites depending on the concentration of phorbol 12, 13 dibutyrate (PDBu) used to activate the response. Translocation of PKCα to the perinucleus but not the plasmalemma was blocked by the use of colchicine to disrupt cell microtubules. However, there was little evidence of significant colocalization of PKCα with the microtubular cytoskeleton during the interval of translocation. By comparison, cytochalasin B disruption of actin microfilaments had no significant effect on PKCα translocation to either the plasmalemma or the perinucleus. The results indicate that the target site of PKCα translocation may vary with activating stimulus strength in A7r5 cells and that the translocation of the isoform to perinuclear target loci depends on an intact microtubular cytoskeleton. This suggests that multiple pathways are available for the redistribution of PKCα that may employ different mechanisms to regulate the movement and/or docking of the isoform at specific target sites.

Concentration-dependent phorbol stimulation of PKCα localization at the nucleus or subplasmalemma in A7r5 cells

AIM: Using confocal microscopy and standard immunohistochemical techniques, changes in the structure of alpha-actin and beta-actin as well as the distribution of myosin II were studied in the contracting A7r5 smooth muscle cell. RESULTS: Prior to stimulation, each of the three proteins were incorporated into filamentous structures extending the length of the cell body. At 30 min after stimulation by phorbol 12, 13 dibutyrate (PDBu), the system of densely packed beta-actin fibres was retained. By comparison, alpha-actin and myosin were observed to undergo significant remodelling, characterized by loss in fibre structure and the formation of brightly fluorescing peripheral bodies. Co-immunoprecipitation of alpha-actin and myosin II suggested an association between the proteins. Consistent with this observation, dual immunostaining for alpha-actin and myosin revealed strong co-localization of the two proteins prior to stimulation. Following PDBu stimulation myosin II was observed to partially disassociate from alpha-actin structure but showed significant co-localization with alpha-actin filaments and peripheral bodies throughout the interval of contraction. The use of cytochalasin B to block actin polymerization or the selective dissolution of alpha-actin cable structure by thapsigargin produced similar patterns of change in alpha-actin structure and the localization of myosin II. CONCLUSION: The results support the concept of myosin liability and potential for remodelling. The results suggest that myosin undergoes extensive relocalization in association with alpha-actin remodelling which may be an important determinant of contractile function in the A7r5 smooth muscle cell.

M. E. Fultz

Papers

PKC‐α shows variable patterns of translocation in response to different stimulatory agents

PKCα translocation is microtubule‐dependent in passaged smooth muscle cells

Concentration-dependent phorbol stimulation of PKCα localization at the nucleus or subplasmalemma in A7r5 cells

Myosin remodelling in the contracting A7r5 smooth muscle cell.