is overexpressed in multiple tumors, leading to the widely held view that this gene drives tumor progression, but this hypothesis has not been rigorously tested in melanoma. Here, we combined a conditional knockout of Coronin 1C with a genetically engineered mouse model of PTEN/ BRAF-driven melanoma. Loss of Coronin 1C in this model increases both primary tumor growth rates and distant metastases. Coronin 1C-null cells isolated from this model are more invasive in vitro and produce more metastatic lesions in orthotopic transplants than Coronin 1C-reexpressing cells due to the shedding of extracellular vesicles (EVs) containing MT1-MMP. Interestingly, these vesicles contain melanosome markers suggesting a melanoma-specific mechanism of EV release, regulated by Coronin 1C, that contributes to the high rates of metastasis in melanoma. Melanoma is the deadliest form of skin cancer with a high propensity for metastatic spread 1,2. The most common genetic drivers of melanoma are BRAF-activating mutations such as V600E, often found in conjunction with loss of tumor suppressors such as PTEN 3-5. If caught in its early stages, melanoma is treatable by surgical resection, but prognosis worsens significantly with the occurrence of in-transit, regional, or distant metastasis. Clinically, up to 54% of metastatic melanoma patients show tumor dissemination to the brain, 77% to the liver, and 85% to the lung 6. The high rate of metastasis in melanoma is poorly understood, but may be linked to lineage-specific factors that impact vesicular trafficking, secretion, degradation, and overall cell migration 7,8. The extracellular matrix (ECM) is an important substrate for tumor cell migration, but it also acts as a physical barrier whose degradation by matrix metalloproteinases (MMPs) is thought to be a critical step in tumor dissemination 9-11. MMPs are a family of both transmembrane (designated "membrane-type" or "MT") and secreted catalytic enzymes capable of cleaving ECM proteins and other substrates. Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a particularly important pro-invasive MMP across many cancer types 12 and whose expression closely correlates with invasion and metastasis 13-15. MT1-MMP is also unique in its ability to both directly cleave a wide range of ECM proteins including collagen types I, II, and III, laminin 1 and 5, and fibronectin, as well as activate pro-MMP2 16. The trafficking of MT1-MMP to and from the plasma membrane (PM) and other membrane structures is surprisingly complex, but critical for the protein's function during tumor invasion and metastasis. Phosphorylation of the cytoplasmic tail initiates internalization of MT1-MMP from the PM by both clathrin-mediated and caveolin-mediated routes 13. Internalized vesicles traffic through the endolysosomal pathway, resulting in redirection to other areas of the PM or to the lysosome for degradation 17. MT1-MMP recycling has been shown to involve various flotilins 18 and SNARE proteins 19-22 , as well as a variety of Rab proteins including Rab5, Rab7 1...

Actin filament dynamics must be precisely controlled in cells to execute behaviors such as vesicular trafficking, cytokinesis, and migration. Coronins are conserved actin-binding proteins that regulate several actin-dependent subcellular processes. Here, we describe a new conditional knockout cell line for two ubiquitous coronins, Coro1B and Coro1C. These coronins, which strongly co-localize with Arp2/3-branched actin, require Arp2/3 activity for proper subcellular localization. Coronin null cells have altered lamellipodial protrusion dynamics due to increased branched actin density and reduced actin turnover within lamellipodia, leading to defective haptotaxis. Surprisingly, excessive cofilin accumulates in coronin null lamellipodia, a result that is inconsistent with the current models of coronin–cofilin functional interaction. However, consistent with coronins playing a pro-cofilin role, coronin null cells have increased F-actin levels. Lastly, we demonstrate that the loss of coronins increases accompanied by an increase in cellular contractility. Together, our observations reveal that coronins are critical for proper turnover of branched actin networks and that decreased actin turnover leads to increased cellular contractility.

Durotaxis, migration of cells directed by a stiffness gradient, is critical in development and disease. To distinguish durotaxis-specific migration mechanisms from those on uniform substrate stiffnesses, we engineered an all-in-one photopolymerized hydrogel system containing areas of stiffness gradients with dual slopes (steep and shallow), adjacent to uniform stiffness (soft and stiff) regions. While fibroblasts rely on nonmuscle myosin II (NMII) activity and the LIM-domain protein Zyxin, ROCK and the Arp2/3 complex are surprisingly dispensable for durotaxis on either stiffness gradient. Additionally, loss of either actin-elongator Formin-like 3 (FMNL3) or actin-bundler fascin has little impact on durotactic response on stiffness gradients. However, lack of Arp2/3 activity results in a filopodia-based durotactic migration that is equally as efficient as that of lamellipodia-based durotactic migration. Importantly, we uncover essential and specific roles for FMNL3 and fascin in the formation and asymmetric distribution of filopodia during filopodia-based durotaxis response to the stiffness gradients. Together, our tunable all-in-one hydrogel system serves to identify both conserved as well as distinct molecular mechanisms that underlie mechano-responses of cells experiencing altered slopes of stiffness gradients.

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Durotaxis, migration of cells directed by stiffness gradient, is critical in development and disease. To study the molecular determinants of single cell durotaxis, we developed an all-in-one photopolymerized hydrogel system containing areas of stiffness gradients with different slopes, along with uniform stiffness (soft and stiff) regions. We find that fibroblasts rely on non-muscle myosin II (NMII) activity and the LIM-domain protein zyxin for durotaxis on both steep and shallow stiffness gradients. Importantly, unlike haptotaxis, the Arp2/3 complex is dispensable for durotaxis on both stiffness gradients. Lack of Arp2/3 results in a filopodia-based durotactic migration that is equally efficient as that of lamellipodia-based durotactic migration. Finally, we reveal an essential role for the actin-bundler fascin in the formation and asymmetric distribution of filopodia during filopodia-based durotaxis in shallow, but not steep, stiffness gradient. Together, our all-in-one hydrogel system can serve as a platform to identify, discriminate, and characterize stiffness gradient specific molecular mechanisms that cells employ to efficiently durotax.