Thin Filament Formation Research Articles

Decay of Taylor–Green flow type initial conditions in a two-dimensional domain

Geophysical flows are often two-dimensional (2D) in nature. The decay dynamics of the canonical, symmetric initial conditions defined by the Taylor–Green (TG) flow is simulated in a 2D domain. The standard lattice Boltzmann equation method with the Bhatnagar–Gross–Krook collision model is employed. The vortex dynamics is investigated for bounded and unbounded flows with various sets of Gaussian vortices and varying Reynolds numbers. The formation of thin filaments and the generation of small-scale structures near the no-slip walls are found. To characterize the decay properties, the integral quantities, namely, kinetic energy, E(t), enstrophy, Ω(t) , and palinstrophy, P(t), are tracked as a function of the eddy-turnover-time. The initial vortex population in the domain dictates the decay rate of these integral quantities. The energy spectra, E(k), are computed to investigate the effect of domain boundaries and vortex populations on energy distribution among different length scales for Re=50000 . The evolution of TG flow in a bounded domain shows an exponent close to −3, which may be attributed to the generation of thin filaments and small-scale structures in vortex-wall interactions. Exponent changes with an increase in the vortex populations.

Ca2+ attenuates nucleation activity of leiomodin.

A transient increase in Ca2+ concentration in sarcomeres is essential for their proper function. Ca2+ drives striated muscle contraction via binding to the troponin complex of the thin filament to activate its interaction with the myosin thick filament. In addition to the troponin complex, the myosin essential light chain and myosin‐binding protein C were also found to be Ca2+ sensitive. However, the effects of Ca2+ on the function of the tropomodulin family proteins involved in regulating thin filament formation have not yet been studied. Leiomodin, a member of the tropomodulin family, is an actin nucleator and thin filament elongator. Using pyrene‐actin polymerization assay and transmission electron microscopy, we show that the actin nucleation activity of leiomodin is attenuated by Ca2+. Using circular dichroism and nuclear magnetic resonance spectroscopy, we demonstrate that the mostly disordered, negatively charged region of leiomodin located between its first two actin‐binding sites binds Ca2+. We propose that Ca2+ binding to leiomodin results in the attenuation of its nucleation activity. Our data provide further evidence regarding the role of Ca2+ as an ultimate regulator of the ensemble of sarcomeric proteins essential for muscle function.Summary StatementCa2+ fluctuations in striated muscle sarcomeres modulate contractile activity via binding to several distinct families of sarcomeric proteins. The effects of Ca2+ on the activity of leiomodin—an actin nucleator and thin filament length regulator—have remained unknown. In this study, we demonstrate that Ca2+ binds directly to leiomodin and attenuates its actin nucleating activity. Our data emphasizes the ultimate role of Ca2+ in the regulation of the sarcomeric protein interactions.

Numb and Numblike regulate sarcomere assembly and maintenance.

A sarcomere is the contractile unit of the myofibril in striated muscles such as cardiac and skeletal muscles. The assembly of sarcomeres depends on multiple molecules that serve as raw materials and participate in the assembly process. However, the mechanism of this critical assembly process remains largely unknown. Here, we found that the cell fate determinant Numb and its homolog Numblike regulated sarcomere assembly and maintenance in striated muscles. We discovered that Numb and Numblike are sarcomeric molecules that were gradually confined to the Z-disc during striated muscle development. Conditional knockout of Numb and Numblike severely compromised sarcomere assembly and its integrity and thus caused organelle dysfunction. Notably, we identified that Numb and Numblike served as sarcomeric α-Actin–binding proteins (ABPs) and shared a conserved domain that can bind to the barbed end of sarcomeric α-Actin. In vitro fluorometric α-Actin polymerization assay showed that Numb and Numblike also played a role in the sarcomeric α-Actin polymerization process. Last, we demonstrate that Numb and Numblike regulate sarcomeric α-Actinin–dependent (ACTN-dependent) Z-disc consolidation in the sarcomere assembly and maintenance. In summary, our studies show that Numb and its homolog Numblike regulate sarcomere assembly and maintenance in striated muscles, and demonstrate a molecular mechanism by which Numb/Numblike, sarcomeric α-Actin, and ACTN cooperate to control thin filament formation and Z-disc consolidation.

Fetal megacystis-microcolon: Genetic mutational spectrum and identification of PDCL3 as a novel candidate gene.

Megacystis-microcolon-intestinal-hypoperistalsis syndrome (MMIHS) is a severe congenital visceral myopathy characterized by an abdominal distension due to a large non-obstructed urinary bladder, a microcolon and intestinal hypo- or aperistalsis. Most of the patients described to date carry a sporadic heterozygous variant in ACTG2. More recently, recessive forms have been reported and mutations in MYH11, LMOD1, MYLK and MYL9 have been described at the molecular level. In the present report, we describe five patients carrying a recurrent heterozygous variant in ACTG2. Exome sequencing performed in four families allowed us to identify the genetic cause in three. In two families, we identified variants in MMIHS causal genes, respectively a nonsense homozygous variant in MYH11 and a previously described homozygous deletion in MYL9. Finally, we identified compound heterozygous variants in a novel candidate gene, PDCL3, c.[143_144del];[380G>A], p.[(Tyr48Ter)];[(Cys127Tyr)]. After cDNA analysis, a complete absence of PDLC3 expression was observed in affected individuals, indicating that both mutated transcripts were unstable and prone to mediated mRNA decay. PDCL3 encodes a protein involved in the folding of actin, a key step in thin filament formation. Presumably, loss-of-function of this protein affects the contractility of smooth muscle tissues, making PDCL3 an excellent candidate gene for autosomal recessive forms of MMIHS.

Dysregulation of NRAP degradation by KLHL41 contributes to pathophysiology in nemaline myopathy.

Nemaline myopathy (NM) is the most common form of congenital myopathy that results in hypotonia and muscle weakness. This disease is clinically and genetically heterogeneous, but three recently discovered genes in NM encode for members of the Kelch family of proteins. Kelch proteins act as substrate-specific adaptors for Cullin 3 (CUL3) E3 ubiquitin ligase to regulate protein turnover through the ubiquitin-proteasome machinery. Defects in thin filament formation and/or stability are key molecular processes that underlie the disease pathology in NM; however, the role of Kelch proteins in these processes in normal and diseases conditions remains elusive. Here, we describe a role of NM causing Kelch protein, KLHL41, in premyofibil-myofibil transition during skeletal muscle development through a regulation of the thin filament chaperone, nebulin-related anchoring protein (NRAP). KLHL41 binds to the thin filament chaperone NRAP and promotes ubiquitination and subsequent degradation of NRAP, a process that is critical for the formation of mature myofibrils. KLHL41 deficiency results in abnormal accumulation of NRAP in muscle cells. NRAP overexpression in transgenic zebrafish resulted in a severe myopathic phenotype and absence of mature myofibrils demonstrating a role in disease pathology. Reducing Nrap levels in KLHL41 deficient zebrafish rescues the structural and function defects associated with disease pathology. We conclude that defects in KLHL41-mediated ubiquitination of sarcomeric proteins contribute to structural and functional deficits in skeletal muscle. These findings further our understanding of how the sarcomere assembly is regulated by disease-causing factors in vivo, which will be imperative for developing mechanism-based specific therapeutic interventions.

CapZ integrates several signaling pathways in response to mechanical stiffness.

Muscle adaptation is a response to physiological demand elicited by changes in mechanical load, hormones, or metabolic stress. Cytoskeletal remodeling processes in many cell types are thought to be primarily regulated by thin filament formation due to actin-binding accessory proteins, such as the actin-capping protein. Here, we hypothesize that in muscle, the actin-capping protein (named CapZ) integrates signaling by a variety of pathways, including phosphorylation and phosphatidylinositol 4,5-bisphosphate (PIP2) binding, to regulate muscle fiber growth in response to mechanical load. To test this hypothesis, we assess mechanotransduction signaling that regulates muscle growth using neonatal rat ventricular myocytes cultured on substrates with the stiffness of the healthy myocardium (10 kPa), fibrotic myocardium (100 kPa), or glass. We investigate how PIP2 signaling affects CapZ using the PIP2 sequestering agent neomycin and the effect of PKC-mediated CapZ phosphorylation using the PKC-activating drug phorbol 12-myristate 13-acetate (PMA). Molecular simulations suggest that close interactions between PIP2 and the β-tentacle of CapZ are modified by phosphorylation at T267. Fluorescence recovery after photobleaching (FRAP) demonstrates that the kinetic binding constant of CapZ to sarcomeric thin filaments in living muscle cells increases with stiffness or PMA treatment but is diminished by PIP2 reduction. Furthermore, CapZ with a deletion of the β-tentacle that lacks the phosphorylation site T267 shows increased FRAP kinetics with lack of sensitivity to PMA treatment or PIP2 reduction. Förster resonance energy transfer (FRET) probes the molecular interactions between PIP2 and CapZ, which are decreased by PIP2 availability or by the β-tentacle truncation. These data suggest that CapZ is bound to actin tightly in the idle, locked state, with little phosphorylation or PIP2 binding. However, this tight binding is loosened in growth states triggered by mechanical stimuli such as substrate stiffness, which may have relevance to fibrotic heart disease.

Leiomodin and Tropomyosin, Binding at the Pointed End of the Thin Filaments

Leiomodin is an important actin-binding protein responsible for the thin filament formation. Mutations in leiomodins were demonstrated to cause myopathies. The spectrum of leiomodin functions remains a subject of an intensive debate. One of the models proposes that leiomodin and its homolog tropomodulin modulate the length of the thin filaments in muscle sarcomeres. The modulation is thought to function through competition between leiomodin and tropomodulin for the pointed end of the thin filaments in a tropomyosin-dependent manner. We studied complexes formed by peptides representing the tropomyosin-binding site of leiomodin 2 (Lmod2s1) and the N-terminal coiled coil fragment of tropomyosin. By using a combination of NMR and MDS methods, we determined the mode of Lmod2s1 binding to the N-terminus of tropomyosin. The structure places leiomodin at the pointed end of thin filaments. This finding provides compelling evidence in favor of the competition hypothesis and has important implications for the role of leiomodin in forming thin filaments in muscle sarcomeres.

Physics and hydraulics of the rhizosphere network

AbstractTake home messageMucilage secreted by roots and EPS produced by microorganisms alter the physical properties of the soil solution and impact the water dynamics in the rhizosphere. The high viscosity of mucilage and EPS is responsible for the formation of thin filaments and interconnected thin lamellae that span throughout the soil matrix maintaining the continuity of the liquid phase across the pore space even during severe drying. The impact of these mechanisms on plant and microorganisms needs to be explored.

Characterization of miRNA and their target gene during chicken embryo skeletal muscle development.

MicroRNAs (miRNAs) are non-coding RNAs that regulate mRNA expression by degradation or translational inhibition. We investigated the underlying molecular mechanisms of skeletal muscle development based on differentially expressed genes and miRNAs. We compared mRNA and miRNA from chicken skeletal muscle at embryonic day E11, E16 and one day post-hatch (P1). The interaction networks were constructed, according to target prediction results and integration analysis of up-regulated genes with down regulated miRNAs or down-regulated genes with up-regulated miRNAs with |log2fold change| ≥ 1.75, P < 0.005. The miRNA-mRNA integration analysis showed high number of mRNAs regulated by a few number of miRNAs. In the E11_VS_E16, comparison group we identified biological processes including muscle maintenance, myoblast proliferation and muscle thin filament formation. The E11_VS_P1 group comparison included negative regulation of axon extension, sarcomere organization, and cell redox homeostasis and kinase inhibitor activity. The E16_VS_P1 comparison group contained genes for the negative regulation of anti-apoptosis and axon extension as well as glomerular basement membrane development. Functional in vitro assays indicated that over expression of miR-222a and miR-126–5p in DF-1 cells significantly reduced the mRNA levels of the target genes CPEB3 and FGFR3, respectively. These integrated analyses provide several candidates for future studies concerning miRNAs-target function on regulation of embryonic muscle development and growth.

DAAM Is Required for Thin Filament Formation and Sarcomerogenesis during Muscle Development in Drosophila

During muscle development, myosin and actin containing filaments assemble into the highly organized sarcomeric structure critical for muscle function. Although sarcomerogenesis clearly involves the de novo formation of actin filaments, this process remained poorly understood. Here we show that mouse and Drosophila members of the DAAM formin family are sarcomere-associated actin assembly factors enriched at the Z-disc and M-band. Analysis of dDAAM mutants revealed a pivotal role in myofibrillogenesis of larval somatic muscles, indirect flight muscles and the heart. We found that loss of dDAAM function results in multiple defects in sarcomere development including thin and thick filament disorganization, Z-disc and M-band formation, and a near complete absence of the myofibrillar lattice. Collectively, our data suggest that dDAAM is required for the initial assembly of thin filaments, and subsequently it promotes filament elongation by assembling short actin polymers that anneal to the pointed end of the growing filaments, and by antagonizing the capping protein Tropomodulin.

Identification of Residues within Tropomodulin-1 Responsible for Its Localization at the Pointed Ends of the Actin Filaments in Cardiac Myocytes

Tropomodulin is a tropomyosin-dependent actin filament capping protein involved in the structural formation of thin filaments and in the regulation of their lengths through its localization at the pointed ends of actin filaments. The disordered N-terminal domain of tropomodulin contains three functional sites: two tropomyosin-binding and one tropomyosin-dependent actin-capping sites. The C-terminal half of tropomodulin consists of one compact domain containing a tropomyosin-independent actin-capping site. Here we determined the structural properties of tropomodulin-1 that affect its roles in cardiomyocytes. To explore the significance of individual tropomyosin-binding sites, GFP-tropomodulin-1 with single mutations that destroy each tropomyosin-binding site was expressed in cardiomyocytes. We demonstrated that both sites are necessary for the optimal localization of tropomodulin-1 at thin filament pointed ends, with site 2 acting as the major determinant. To investigate the functional properties of the tropomodulin C-terminal domain, truncated versions of GFP-tropomodulin-1 were expressed in cardiomyocytes. We discovered that the leucine-rich repeat (LRR) fold and the C-terminal helix are required for its proper targeting to the pointed ends. To investigate the structural significance of the LRR fold, we generated three mutations within the C-terminal domain (V232D, F263D, and L313D). Our results show that these mutations affect both tropomyosin-independent actin-capping activity and pointed end localization, most likely by changing local conformations of either loops or side chains of the surfaces involved in the interactions of the LRR domain. Studying the influence of these mutations individually, we concluded that, in addition to the tropomyosin-independent actin-capping site, there appears to be another regulatory site within the tropomodulin C-terminal domain.

Tropomyosin assembly intermediates in the control of microfilament system turnover

Tropomyosin is a coiled-coil α-helical protein, which self-associates in a head-to-tail fashion along polymers of actin to produce thin filaments. Mammalian non-muscle cells express a large number of tropomyosin isoforms, which are differentially regulated during embryogenesis and associated with specialized actin microfilament ensembles in cells. The function of tropomyosin in specifying form and localization of these subcellular structures, and the precise mechanism(s) by which they carry out their functions, is unclear. This paper reports that, while the major fraction of non-muscle cell tropomyosin resides in actin thin filaments of the cytomatrix, the soluble part of the cytoplasm contains tropomyosins in the form of actin-free multimers, which are isoform specific and of high molecular weight (MW app 180,000–250,000). Stimulation of motile cells with growth factors induces a rapid, actin polymerization-dependent outgrowth of lamellipodia and filopodia. Concomitantly, the levels of tropomyosin isoform-specific multimers decrease, suggesting their involvement in actin thin filament formation. Malignant tumor cells have drastically altered levels and composition of tropomyosin isoform-specific multimers as well as tropomyosin in the cytomatrix.

Ectopic expression and dynamics of TPM1α and TPM1κ in myofibrils of avian myotubes

From the four known vertebrate tropomyosin genes (designated TPM1, TPM2, TPM3, and TPM4) over 20 isoforms can be generated. The predominant TPM1 isoform, TPM1alpha, is specifically expressed in both skeletal and cardiac muscles. A newly discovered alternatively spliced isoform, TPM1kappa, containing exon 2a instead of exon 2b contained in TPM1alpha, was found to be cardiac specific and developmentally regulated. In this work, we transfected quail skeletal muscle cells with green fluorescent proteins (GFP) coupled to chicken TPM1alpha and chicken TPM1kappa and compared their localizations in premyofibrils and mature myofibrils. We used the technique of fluorescence recovery after photobleaching (FRAP) to compare the dynamics of TPM1alpha and TPM1kappa in myotubes. TPM1alpha and TPM1kappa incorporated into premyofibrils, nascent myofibrils, and mature myofibrils of quail myotubes in identical patterns. The two tropomyosin isoforms have a higher exchange rate in premyofibrils than in mature myofibrils. F-actin and muscle tropomyosin are present in the same fibers at all three stages of myofibrillogenesis (premyofibrils, nascent myofibrils, mature myofibrils). In contrast, the tropomyosin-binding molecule nebulin is not present in the initial premyofibrils. Nebulin is gradually added during myofibrillogenesis, becoming fully localized in striated patterns by the mature myofibril stage. A model of thin filament formation is proposed to explain the increased stability of tropomyosin in mature myofibrils. These experiments are supportive of a maturing thin filament and stepwise model of myofibrillogenesis (premyofibrils to nascent myofibrils to mature myofibrils), and are inconsistent with models that postulate the immediate appearance of fully formed thin filaments or myofibrils.

Structural transition of the inhibitory region of troponin I within the regulated cardiac thin filament

Contraction and relaxation of cardiac muscle are regulated by the inhibitory and regulatory regions of troponin I (cTnI). Our previous FRET studies showed that the inhibitory region of cTnI in isolated troponin experiences a structural transition from a β-turn/coil motif to an extended conformation upon Ca 2+ activation. During the relaxation process, the kinetics of the reversal of this conformation is coupled to the closing of the Ca 2+-induced open conformation of the N-domain of troponin C (cTnC) and an interaction between cTnC and cTnI in their interface. We have since extended the structural kinetic study of the inhibitory region to fully regulated thin filament. Single-tryptophan and single-cysteine mutant cTnI(L129W/S151C) was labeled with 1,5-IAEDANS at Cys151, and the tryptophan-AEDANS pair served as a donor–acceptor pair. Labeled cTnI mutant was used to prepare regulated thin filaments. Ca 2+-induced conformational changes in the segment of Trp129-Cys151 of cTnI were monitored by FRET sensitized acceptor (AEDANS) emission in Ca 2+ titration and stopped-flow measurements. Control experiments suggested energy transfer from endogenous tryptophan residues of actin and myosin S1 to AEDANS attached to Cys151 of cTnI was very small and Ca 2+ independent. The present results show that the rate of Ca 2+-induced structural transition and Ca 2+ sensitivity of the inhibitory region of cTnI were modified by (1) thin filament formation, (2) the presence of strongly bound S1, and (3) PKA phosphorylation of the N-terminus of cTnI. Ca 2+ sensitivity was not significantly changed by the presence of cTm and actin. However, the cTn–cTm interaction decreased the cooperativity and kinetics of the structural transition within cTnI, while actin filaments elicited opposite effects. The strongly bound S1 significantly increased the Ca 2+ sensitivity and slowed down the kinetics of structural transition. In contrast, PKA phosphorylation of cTnI decreased the Ca 2+ sensitivity and accelerated the structural transition rate of the inhibitory region of cTnI on thin filaments. These results support the idea of a feedback mechanism by strong cross-bridge interaction with actin and provide insights on the molecular basis for the fine tuning of cardiac function by β-adrenergic stimulation.

Electron microscopic study of actin polymerization in airway smooth muscle.

Actin polymerization as part of the normal smooth muscle response to various stimuli has been reported. The actin dynamics are believed to be necessary for cytoskeletal remodeling in smooth muscle in its adaptation to external stress and strain and for maintenance of optimal contractility. We have shown in our previous studies in airway smooth muscle that myosins polymerized in response to contractile activation as well as to adaptation at longer cell lengths. We postulated that the same response could be elicited from actins under the same conditions. In the present study, actin filament formation was quantified electron microscopically in cell cross sections. Nanometer resolution allowed us to examine regional distribution of filaments in a cell cross section. Airway smooth muscle bundles were fixed in relaxed and activated states at two lengths; muscle preparations were also fixed after a period of oscillatory strain, a condition known to cause depolymerization of myosin filaments. The results indicate that contractile activation and increased cell length nonsynergistically enhanced actin polymerization; the extent of actin polymerization was substantially less than that of myosin polymerization. Oscillatory strain increased thin filament formation. Although thin filament density was found higher in cytoplasmic areas near dense bodies, contractile activation did not preferentially enhance actin polymerization in these areas. It is concluded that actin thin filaments are dynamic structures whose length and number are regulated by the cell in response to changes in extracellular environment and that polymerization and depolymerization of thin filaments occur uniformly across the whole cell cross section.

Calcium transients regulate patterned actin assembly during myofibrillogenesis.

The highly ordered arrangement of sarcomeric myosin during striated muscle development requires spontaneous calcium (Ca(2+)) transients. Here, we show that blocking transients also compromises patterned assembly of actin thin filaments, titin, and capZ. Because a conserved temporal assembly pattern has been described for these proteins, selective inhibitors of either thick or thin filament formation were used to determine their relative temporal interdependencies. For example, inhibition of myosin light chain kinase (MLCK) by application of a specific inhibitory peptide or phorbol myistate acetate (PMA) disrupts myosin assembly without significantly affecting formation of actin bands. The MLCK inhibitor ML-7, however, disrupted actin as well as myosin. Surprisingly, agents that interfere with actin dynamics, such as cytochalasin D, produced only minor organizational disruptions in actin, capZ, and titin staining. However, cytochalasin D and other actin disrupting compounds significantly perturbed myosin organization. The results indicate that (1) Ca(2+) transients regulate one or more of the earliest steps in sarcomere formation, (2) mature actin filaments can assemble independently of myosin band formation, and (3) myosin thick filament assembly is extremely sensitive to disruption of either the actin or titin filament systems.

Nemaline Myopathy Caused by Mutations in the Muscle α-Skeletal-Actin Gene

Nemaline myopathy (NM) is a clinically and genetically heterogeneous disorder characterized by muscle weakness and the presence of nemaline bodies (rods) in skeletal muscle. Disease-causing mutations have been reported in five genes, each encoding a protein component of the sarcomeric thin filament. Recently, we identified mutations in the muscle alpha-skeletal-actin gene (ACTA1) in a subset of patients with NM. In the present study, we evaluated a new series of 35 patients with NM. We identified five novel missense mutations in ACTA1, which suggested that mutations in muscle alpha-skeletal actin account for the disease in approximately 15% of patients with NM. The mutations appeared de novo and represent new dominant mutations. One proband subsequently had two affected children, a result consistent with autosomal dominant transmission. The seven patients exhibited marked clinical variability, ranging from severe congenital-onset weakness, with death from respiratory failure during the 1st year of life, to a mild childhood-onset myopathy, with survival into adulthood. There was marked variation in both age at onset and clinical severity in the three affected members of one family. Common pathological features included abnormal fiber type differentiation, glycogen accumulation, myofibrillar disruption, and "whorling" of actin thin filaments. The percentage of fibers with rods did not correlate with clinical severity; however, the severe, lethal phenotype was associated with both severe, generalized disorganization of sarcomeric structure and abnormal localization of sarcomeric actin. The marked variability, in clinical phenotype, among patients with different mutations in ACTA1 suggests that both the site of the mutation and the nature of the amino acid change have differential effects on thin-filament formation and protein-protein interactions. The intrafamilial variability suggests that alpha-actin genotype is not the sole determinant of phenotype.

Interfacial instabilities of a fluid annulus in a rotating Hele–Shaw cell

We have studied the interfacial instabilities experienced by a liquid annulus as it moves radially in a circular Hele–Shaw cell rotating with angular velocity Ω. The instability of the leading interface (oil displacing air) is driven by the density difference in the presence of centrifugal forcing, while the instability of the trailing interface (air displacing oil) is driven by the large viscosity contrast. A linear stability analysis shows that the stability of the two interfaces is coupled through the pressure field already at a linear level. We have performed experiments in a dry cell and in a cell coated with a thin fluid layer on each plate, and found that the stability depends substantially on the wetting conditions at the leading interface. Our experimental results of the number of fingers resulting from the instability compare well with the predictions obtained through a numerical integration of the coupled equations derived from a linear stability analysis. Deep in the nonlinear regime we observe the emission of liquid droplets through the formation of thin filaments at the tip of outgrowing fingers.

Evolution of a Potential Vorticity Front over a Topographic Slope

Using the methodology of contour dynamics, the evolution of an interface separating two regions of constant potential vorticity is considered. The model equations are those for barotropic nondivergent flow over a topographic slope adjoined by a coastal barrier. The main focus is on the processes which lead to wave breaking for small-amplitude waves and, in general agreement with previous related work, it is found that wave steepening leads to the formation of thin filaments. The key parameter is found to be the ratio of the background potential vorticity to the discontinuity of potential vorticity across the front, with other parameters such as the topographic slope and the distance to the coast playing a relatively less significant role.