mRNA location and translation rate determine protein targeting to dual destinations.

Numerous proteins are targeted to two or multiple subcellular destinations where they exert distinct functional consequences. The balance between such differential targeting is thought to be determined post-translationally, relying on protein sorting mechanisms. Here, we show that mRNA location and translation rate can also determine protein targeting by modulating protein binding to specific interacting partners. Peripheral localization of the NET1 mRNA and fast translation lead to higher cytosolic retention of the NET1 protein by promoting its binding to the membrane-associated scaffold protein CASK. By contrast, perinuclear mRNA location and/or slower translation rate favor nuclear targeting by promoting binding to importins. This mRNA location-dependent mechanism is modulated by physiological stimuli and profoundly impacts NET1 function in cell motility. These results reveal that the location of protein synthesis and the rate of translation elongation act in coordination as a "partner-selection" mechanism that robustly influences protein distribution and function.

RNA localization and co-translational interactions control RAB13 GTPase function and cell migration.

Numerous RNAs exhibit specific distribution patterns in mammalian cells. However, the functional and mechanistic consequences are relatively unknown. Here, we investigate the functional role of RNA localization at cellular protrusions of migrating mesenchymal cells, using as a model the RAB13 RNA, which encodes a GTPase important for vesicle-mediated membrane trafficking. While RAB13 RNA is enriched at peripheral protrusions, the expressed protein is concentrated perinuclearly. By specifically preventing RAB13 RNA localization, we show that peripheral RAB13 translation is not important for the overall distribution of the RAB13 protein or its ability to associate with membranes, but is required for full activation of the GTPase and for efficient cell migration. RAB13 translation leads to a co-translational association of nascent RAB13 with the exchange factor RABIF. Our results indicate that RAB13-RABIF association at the periphery is required for directing RAB13 GTPase activity to promote cell migration. Thus, translation of RAB13 in specific subcellular environments imparts the protein with distinct properties and highlights a means of controlling protein function through local RNA translation.

Collective cancer cell invasion requires RNA accumulation at the invasive front.

Localization of RNAs at protrusive regions of cells is important for single-cell migration on two-dimensional surfaces. Protrusion-enriched RNAs encode factors linked to cancer progression, such as the RAB13 GTPase and the NET1 guanine nucleotide exchange factor, and are regulated by the tumor-suppressor protein APC. However, tumor cells in vivo often do not move as single cells but rather utilize collective modes of invasion and dissemination. Here, we developed an inducible system of three-dimensional (3D) collective invasion to study the behavior and importance of protrusion-enriched RNAs. We find that, strikingly, both the RAB13 and NET1 RNAs are enriched specifically at the invasive front of leader cells in invasive cell strands. This localization requires microtubules and coincides with sites of high laminin concentration. Indeed, laminin association and integrin engagement are required for RNA accumulation at the invasive front. Importantly, perturbing RNA accumulation reduces collective 3D invasion. Examination of in vivo tumors reveals a similar localization of the RAB13 and NET1 RNAs at potential invasive sites, suggesting that this mechanism could provide a targeting opportunity for interfering with collective cancer cell invasion.

Transient mechanical strain promotes the maturation of invadopodia and enhances cancer cell invasion in vitro.

Cancer cell invasion is influenced by various biomechanical forces found within the microenvironment. We have previously found that invasion is enhanced in fibrosarcoma cells when transient mechanical stimulation is applied within an in vitro mechano-invasion assay. This enhancement of invasion is dependent on cofilin (CFL1), a known regulator of invadopodia maturation. Invadopodia are actin-rich structures present in invasive cancer cells that are enzymatically active and degrade the surrounding extracellular matrix to facilitate invasion. In this study, we examine changes in gene expression in response to tugging on matrix fibers. Interestingly, we find that integrin ß3 expression is downregulated and leads to an increase in cofilin activity, as evidenced by a reduction in its Ser3 phosphorylation levels. As a result, invadopodia lengthen and have increased enzymatic activity, indicating that transient mechanical stimulation promotes the maturation of invadopodia leading to increased levels of cell invasion. Our results are unique in defining an invasive mechanism specific to the invasive process of cancer cells that is triggered by tugging forces in the microenvironment, as opposed to rigidity, compression or stretch forces.

Cardiolipin Regulates Mitophagy through the Protein Kinase C Pathway.

Cardiolipin (CL), the signature phospholipid of mitochondrial membranes, is important for cardiovascular health, and perturbation of CL metabolism is implicated in cardiovascular disease. Although the role of CL in mitochondrial function, biogenesis, and genome stability has been studied, recent findings indicate that it is essential for functions apart from mitochondrial bioenergetics. In this study, we report that mitophagy is perturbed in CL-deficient yeast cells. Mutants of autophagy/mitophagy genes ATG8, ATG18, and ATG32 synthetically interact with CL synthase mutant crd1Δ. CL-deficient cells exhibited decreased GFP-tagged mitochondrial proteins inside the vacuole and decreased free GFP, consistent with decreased mitophagy. Both PKC and high osmolarity glycerol (HOG) MAPK pathways were shown previously to be required for mitophagy. Activation of both MAPKs was defective in CL-deficient cells. Deletion of HOG pathway genes SHO1, SSK1, STE50, and HOG1 exacerbated crd1Δ growth. 1 m sorbitol and 0.2 m NaCl, which induce the HOG pathway, rescued growth of the mutant. Activation of the MAPK Slt2p was defective in crd1Δ cells, and up-regulation of the PKC pathway by expression of the PKC1R398P gene, which encodes constitutively activated Pkc1p, rescued crd1Δ growth and mitophagy defects. These findings indicate that loss of CL impairs MAPK pathway activation, and decreased activation of the PKC pathway leads to defective mitophagy.

Mechanoreception at the cell membrane: More than the integrins.

A cell receives mechanical cues from its surrounding microenvironment and transduces this mechanical information into a biochemical signal within the cell, ultimately resulting in physiological change. Several molecules within the plasma membrane have been identified that are capable of receiving and translating a mechanical signal. Although integrins are most often discussed as the cell's primary method of mechanoreception at the cell membrane, several non-integrin mechanoreceptors have emerged over the last decade. Specifically, multiple G-protein coupled receptors, the glycocalyx, ion channels, lipid rafts and receptor tyrosine kinases have been found to translate mechanical stimuli from the environment into cellular change. This review will discuss these non-integrin mechanoreceptors associated with the plasma membrane, and their impact on cell physiology.

A KIF1C-CNBP motor-adaptor complex for trafficking mRNAs to cell protrusions.

mRNA localization to subcellular compartments is a widely used mechanism that functionally contributes to numerous processes. mRNA targeting can be achieved upon recognition of RNA cargo by molecular motors. However, our molecular understanding of how this is accomplished is limited, especially in higher organisms. We focus on a pathway that targets mRNAs to peripheral protrusions of mammalian cells and is important for cell migration. Trafficking occurs through active transport on microtubules, mediated by the KIF1C kinesin. Here, we identify the RNA-binding protein CNBP, as a factor required for mRNA localization to protrusions. CNBP binds directly to GA-rich sequences in the 3'UTR of protrusion targeted mRNAs. CNBP also interacts with KIF1C and is required for KIF1C recruitment to mRNAs and for their trafficking on microtubules to the periphery. This work provides a molecular mechanism for KIF1C recruitment to mRNA cargo and reveals a motor-adaptor complex for mRNA transport to cell protrusions.

mRNA Location and Translation Rate Determine Protein Targeting to Dual Destinations.

Numerous proteins are targeted to two or multiple subcellular destinations where they exert distinct functional consequences. The balance between such differential targeting is thought to be determined post-translationally, relying on protein sorting mechanisms. Here, we show that protein targeting can additionally be determined by mRNA location and translation rate, through modulating protein binding to specific interacting partners. Peripheral localization of the NET1 mRNA and fast translation lead to higher cytosolic retention of the NET1 protein, through promoting its binding to the membrane-associated scaffold protein CASK. By contrast, perinuclear mRNA location and/or slower translation rate favor nuclear targeting, through promoting binding to importins. This mRNA location-dependent mechanism is modulated by physiological stimuli and profoundly impacts NET1 function in cell motility. These results reveal that the location of protein synthesis and the rate of translation elongation act in coordination as a 'partner-selection' mechanism that robustly influences protein distribution and function.

Regulation and outcomes of localized RNA translation.

Spatial segregation of mRNAs in the cytoplasm of cells is a well-known biological phenomenon that is widely observed in diverse species spanning different kingdoms of life. In mammalian cells, localization of mRNAs has been documented and studied quite extensively in highly polarized cells, most notably in neurons, where localized mRNAs function to direct protein production at sites that are quite distant from the soma. Recent studies have strikingly revealed that a large proportion of the cellular transcriptome exhibits polarized distributions even in cells that lack an obvious need for long-range transport, such as fibroblasts or epithelial cells. This review focuses on emerging concepts regarding the functional outcomes of mRNA targeting in the cytoplasm of such cells. We also discuss regulatory mechanisms controlling these events, with an emphasis on the role of cell mechanics and the organization of the cytoskeleton. This article is categorized under: Translation > Regulation RNA Export and Localization > RNA Localization.

The Role of PAK1 in the Maturation of Invadopodia During Transient Mechanical Stimulation.

Cancer cells are affected by a wide range of mechanical forces within their extracellular environment. It has been widely shown that these forces can lead to increased metastatic activity of these cells. One such force is a transient tugging-like force that results from contractile forces generated by cells within the tumor microenvironment. When this force is simulated in vitro with a mechano-invasion assay, human fibrosarcoma cells exhibit enhanced cell invasion in a 3D collagen-fibronectin matrix by downregulating the expression of integrin ß3. Furthermore, this force stimulates the maturation of invadopodia in an integrin ß3-dependent manner that includes an increase in the active form of cofilin and MMP-2 secretion. In the present study we discovered that the decrease in integrin ß3 signaling in response to mechanical stimulation is coupled to the activity of p21-activated kinase 1 (PAK1). It was found that PAK1 has decreased activity, as detected by a decrease in Ser144 phosphorylation, with mechanical stimulation. However, this loss in phosphorylation can be reversed if integrin ß3 is overexpressed. Furthermore, PAK1 mutants show a correlated response in MMP-2 enzyme expression and activity, in addition to the lengthening of invadopodia, in response to stimulation. These results identify a novel mechano-sensitive response in human fibrosarcoma that utilizes PAK1 as a signaling player positioned downstream of integrin ß3.

An in vitro correlation of metastatic capacity and dual mechanostimulation.

Cells are under the influence of multiple forms of mechanical stimulation in vivo. For example, a cell is subjected to mechanical forces from tissue stiffness, shear and tensile stress and transient applied strain. Significant progress has been made in understanding the cellular mechanotransduction mechanisms in response to a single mechanical parameter. However, our knowledge of how a cell responds to multiple mechanical inputs is currently limited. In this study, we have tested the cellular response to the simultaneous application of two mechanical inputs: substrate compliance and transient tugging. Our results suggest that cells within a multicellular spheroid will restrict their response to a single mechanical input at a time and when provided with two mechanical inputs simultaneously, one will dominate. In normal and non-metastatic mammary epithelial cells, we found that they respond to applied stimulation and will override substrate compliance cues in favor of the applied mechanical stimulus. Surprisingly, however, metastatic mammary epithelial cells remain non-responsive to both mechanical cues. Our results suggest that, within our assay system, metastatic progression may involve the down-regulation of multiple mechanotransduction pathways.