Multiplexed imaging to reveal tissue dendritic cell spatial localisation and function.

Dendritic cells (DCs) play a pivotal role in immune surveillance, acting as sentinels that coordinate immune responses within tissues. Although differences in the identity and functional states of DC subpopulations have been identified through multiparametric flow cytometry and single-cell RNA sequencing, these methods do not provide information about the spatial context in which the cells are located. This knowledge is crucial for understanding tissue organisation and cellular cross-talk. Recent developments in multiplex imaging techniques can now offer insights into this complex spatial and functional landscape. This review provides a concise overview of these imaging methodologies, emphasising their application in identifying DCs to delineate their tissue-specific functions and aiding newcomers in navigating this field.

Aberrant cortex contractions impact mammalian oocyte quality.

The cortex controls cell shape. In mouse oocytes, the cortex thickens in an Arp2/3-complex-dependent manner, ensuring chromosome positioning and segregation. Surprisingly, we identify that mouse oocytes lacking the Arp2/3 complex undergo cortical actin remodeling upon division, followed by cortical contractions that are unprecedented in mammalian oocytes. Using genetics, imaging, and machine learning, we show that these contractions stir the cytoplasm, resulting in impaired organelle organization and activity. Oocyte capacity to avoid polyspermy is impacted, leading to a reduced female fertility. We could diminish contractions and rescue cytoplasmic anomalies. Similar contractions were observed in human oocytes collected as byproducts during IVF (in vitro fertilization) procedures. These contractions correlate with increased cytoplasmic motion, but not with defects in spindle assembly or aneuploidy in mice or humans. Our study highlights a multiscale effect connecting cortical F-actin, contractions, and cytoplasmic organization and affecting oocyte quality, with implications for female fertility.

Investigating Mammalian Formins with SMIFH2 Fifteen Years in: Novel Targets and Unexpected Biology.

The mammalian formin family comprises fifteen multi-domain proteins that regulate actin dynamics and microtubules in vitro and in cells. Evolutionarily conserved formin homology (FH) 1 and 2 domains allow formins to locally modulate the cell cytoskeleton. Formins are involved in several developmental and homeostatic processes, as well as human diseases. However, functional redundancy has long hampered studies of individual formins with genetic loss-of-function approaches and prevents the rapid inhibition of formin activities in cells. The discovery of small molecule inhibitor of formin homology 2 domains (SMIFH2) in 2009 was a disruptive change that provided a powerful chemical tool to explore formins' functions across biological scales. Here, I critically discuss the characterization of SMIFH2 as a pan-formin inhibitor, as well as growing evidence of unexpected off-target effects. By collating the literature and information hidden in public repositories, outstanding controversies and fundamental open questions about the substrates and mechanism of action of SMIFH2 emerge. Whenever possible, I propose explanations for these discrepancies and roadmaps to address the paramount open questions. Furthermore, I suggest that SMIFH2 be reclassified as a multi-target inhibitor for its appealing activities on proteins involved in pathological formin-dependent processes. Notwithstanding all drawbacks and limitations, SMIFH2 will continue to prove useful in studying formins in health and disease in the years to come.

Advanced Cell Culture Models Illuminate the Interplay between Mammary Tumor Cells and Activated Fibroblasts.

The interaction between tumor cells and activated fibroblasts determines malignant features of desmoplastic carcinomas such as rapid growth, progression towards a metastatic phenotype, and resistance to chemotherapy. On one hand, tumor cells can activate normal fibroblasts and even reprogram them into CAFs through complex mechanisms that also involve soluble factors. Among them, transforming growth factor beta (TGF-ß) and Platelet-Derived Growth Factor (PDGF) have an established role in the acquisition of pro-tumorigenic phenotypes by fibroblasts. On the other hand, activated fibroblasts release Interleukin-6 (IL-6), which increases tumor-cell invasiveness and chemoresistance. However, the interplay between breast cancer cells and fibroblasts, as well as the modes of action of TGF-ß, PDGF, and IL-6, are difficult to investigate in vivo. Here, we validated the usage of advanced cell culture models as tools to study the interplay between mammary tumor cells and fibroblasts, taking mouse and human triple-negative tumor cells and fibroblasts as a case study. We employed two different settings, one permitting only paracrine signaling, the other both paracrine and cell-contact-based signaling. These co-culture systems allowed us to unmask how TGF-ß, PDGF and IL-6 mediate the interplay between mammary tumor cells and fibroblasts. We found that the fibroblasts underwent activation induced by the TGF-ß and the PDGF produced by the tumor cells, which increased their proliferation and IL-6 secretion. The IL-6 secreted by activated fibroblasts enhanced tumor-cell proliferation and chemoresistance. These results show that these breast cancer avatars possess an unexpected high level of complexity, which resembles that observed in vivo. As such, advanced co-cultures provide a pathologically relevant tractable system to study the role of the TME in breast cancer progression with a reductionist approach.

A Protumorigenic mDia2-MIRO1 Axis Controls Mitochondrial Positioning and Function in Cancer-Associated Fibroblasts.

Cancer-associated fibroblasts (CAF) are key regulators of tumorigenesis. Further insights into the tumor-promoting mechanisms of action of CAFs could help improve cancer diagnosis and treatment. Here we show that the formin mDia2 regulates the positioning and function of mitochondria in dermal fibroblasts, thereby promoting a protumorigenic CAF phenotype. Mechanistically, mDia2 stabilized the mitochondrial trafficking protein MIRO1. Loss of mDia2 or MIRO1 in fibroblasts or CAFs reduced the presence of mitochondria and ATP levels near the plasma membrane and at CAF-tumor cell contact sites, caused metabolic alterations characteristic of mitochondrial dysfunction, and suppressed the secretion of protumorigenic proteins. In mouse models of squamous carcinogenesis, genetic or pharmacologic inhibition of mDia2, MIRO1, or their common upstream regulator activin A inhibited tumor formation. Consistently, co-upregulation of mDia2 and MIRO1 in the stroma of various human cancers negatively correlated with survival. This work unveils a key role of mitochondria in the protumorigenic CAF phenotype and identifies an activin A-mDia2-MIRO1 signaling axis in CAFs with diagnostic and therapeutic potential. SIGNIFICANCE: Inhibition of mDia2/MIRO1-mediated mitochondrial positioning in CAFs induces mitochondrial dysfunction and suppresses tumor growth, revealing a promising therapeutic strategy to target tumor-stroma cross-talk.

mDia1 Assembles a Linear F-Actin Coat at Membrane Invaginations To Drive Listeria monocytogenes Cell-to-Cell Spreading.

Direct cell-to-cell spreading of Listeria monocytogenes requires the bacteria to induce actin-based finger-like membrane protrusions in donor host cells that are endocytosed through caveolin-rich membrane invaginations by adjacent receiving cells. An actin shell surrounds these endocytic sites; however, its structure, composition, and functional significance remain elusive. Here, we show that the formin mDia1, but surprisingly not the Arp2/3 complex, is enriched at the membrane invaginations generated by L. monocytogenes during HeLa and Jeg-3 cell infections. Electron microscopy reveals a band of linear actin filaments that run along the longitudinal axis of the invagination membrane. Mechanistically, mDia1 expression is vital for the assembly of this F-actin shell. mDia1 is also required for the recruitment of Filamin A, a caveola-associated F-actin cross-linking protein, and caveolin-1 to the invaginations. Importantly, mixed-cell infection assays show that optimal caveolin-based L. monocytogenes cell-to-cell spreading correlates with the formation of the linear actin filament-containing shell by mDia1. IMPORTANCE Listeria monocytogenes spreads from one cell to another to colonize tissues. This cell-to-cell movement requires the propulsive force of an actin-rich comet tail behind the advancing bacterium, which ultimately distends the host plasma membrane into a slender bacterium-containing membrane protrusion. These membrane protrusions induce a corresponding invagination in the membrane of the adjacent host cell. The host cell that receives the protrusion utilizes caveolin-based endocytosis to internalize the structures, and filamentous actin lines these membrane invaginations. Here, we set out to determine the structure and function of this filamentous actin "shell." We demonstrate that the formin mDia1, but not the Arp2/3 complex, localizes to the invaginations. Morphologically, we show that this actin is organized into linear arrays and not branched dendritic networks. Mechanistically, we show that the actin shell is assembled by mDia1 and that mDia1 is required for efficient cell-to-cell transfer of L. monocytogenes.

mTORC1 and mTORC2 Converge on the Arp2/3 Complex to Promote KrasG12D-Induced Acinar-to-Ductal Metaplasia and Early Pancreatic Carcinogenesis.

BACKGROUND & AIMS: Oncogenic KrasG12D induces neoplastic transformation of pancreatic acinar cells through acinar-to-ductal metaplasia (ADM), an actin-based morphogenetic process, and drives pancreatic ductal adenocarcinoma (PDAC). mTOR (mechanistic target of rapamycin kinase) complex 1 (mTORC1) and 2 (mTORC2) contain Rptor and Rictor, respectively, and are activated downstream of KrasG12D, thereby contributing to PDAC. Yet, whether and how mTORC1 and mTORC2 impact on ADM and the identity of the actin nucleator(s) mediating such actin rearrangements remain unknown. METHODS: A mouse model of inflammation-accelerated KrasG12D-driven early pancreatic carcinogenesis was used. Rptor, Rictor, and Arpc4 (actin-related protein 2/3 complex subunit 4) were conditionally ablated in acinar cells to deactivate the function of mTORC1, mTORC2 and the actin-related protein (Arp) 2/3 complex, respectively. RESULTS: We found that mTORC1 and mTORC2 are markedly activated in human and mouse ADM lesions, and cooperate to promote KrasG12D-driven ADM in mice and in vitro. They use the Arp2/3 complex as a common downstream effector to induce the remodeling the actin cytoskeleton leading to ADM. In particular, mTORC1 regulates the translation of Rac1 (Rac family small GTPase 1) and the Arp2/3-complex subunit Arp3, whereas mTORC2 activates the Arp2/3 complex by promoting Akt/Rac1 signaling. Consistently, genetic ablation of the Arp2/3 complex prevents KrasG12D-driven ADM in vivo. In acinar cells, the Arp2/3 complex and its actin-nucleation activity mediated the formation of a basolateral actin cortex, which is indispensable for ADM and pre-neoplastic transformation. CONCLUSIONS: Here, we show that mTORC1 and mTORC2 attain a dual, yet nonredundant regulatory role in ADM and early pancreatic carcinogenesis by promoting Arp2/3 complex function. The role of Arp2/3 complex as a common effector of mTORC1 and mTORC2 fills the gap between oncogenic signals and actin dynamics underlying PDAC initiation.

The chloride intracellular channel protein CLIC4 inhibits filopodium formation induced by constitutively active mutants of formin mDia2.

Chloride intracellular channel 4 (CLIC4) functions in diverse actin-dependent processes. Upon Rho activation, CLIC4 reversibly translocates from the cytosol to the plasma membrane to regulate cell adhesion and migration. At the plasma membrane, CLIC4 counters the formation of filopodia, which requires actin assembly by the formin mammalian Diaphanous (mDia)2. To this end, mDia2 must be activated through conversion from the closed to the open conformation. Thus, CLIC4 could harness the activation or the open conformation of mDia2 to inhibit filopodium formation. Here, we find that CLIC4 silencing enhances the filopodia induced by two constitutively active mDia2 mutants. Furthermore, we report that CLIC4 binds the actin-regulatory region of mDia2 in vitro. These results suggest that CLIC4 modulates the activity of the open conformation of mDia2, shedding new light into how cells may control filopodia.

A paracrine activin A-mDia2 axis promotes squamous carcinogenesis via fibroblast reprogramming.

Cancer-associated fibroblasts (CAFs) are key regulators of tumorigenesis and promising targets for next-generation therapies. We discovered that cancer cell-derived activin A reprograms fibroblasts into pro-tumorigenic CAFs. Mechanistically, this occurs via Smad2-mediated transcriptional regulation of the formin mDia2, which directly promotes filopodia formation and cell migration. mDia2 also induces expression of CAF marker genes through prevention of p53 nuclear accumulation, resulting in the production of a pro-tumorigenic matrisome and secretome. The translational relevance of this finding is reflected by activin A overexpression in tumor cells and of mDia2 in the stroma of skin cancer and other malignancies and the correlation of high activin A/mDia2 levels with poor patient survival. Blockade of this signaling axis using inhibitors of activin, activin receptors, or mDia2 suppressed cancer cell malignancy and squamous carcinogenesis in 3D organotypic cultures, ex vivo, and in vivo, providing a rationale for pharmacological inhibition of activin A-mDia2 signaling in stratified cancer patients.

Profilin binding couples chloride intracellular channel protein CLIC4 to RhoA-mDia2 signaling and filopodium formation.

Chloride intracellular channel 4 (CLIC4) is a cytosolic protein implicated in diverse actin-based processes, including integrin trafficking, cell adhesion, and tubulogenesis. CLIC4 is rapidly recruited to the plasma membrane by RhoA-activating agonists and then partly colocalizes with ß1 integrins. Agonist-induced CLIC4 translocation depends on actin polymerization and requires conserved residues that make up a putative binding groove. However, the mechanism and significance of CLIC4 trafficking have been elusive. Here, we show that RhoA activation by either lysophosphatidic acid (LPA) or epidermal growth factor is necessary and sufficient for CLIC4 translocation to the plasma membrane and involves regulation by the RhoA effector mDia2, a driver of actin polymerization and filopodium formation. We found that CLIC4 binds the G-actin-binding protein profilin-1 via the same residues that are required for CLIC4 trafficking. Consistently, shRNA-induced profilin-1 silencing impaired agonist-induced CLIC4 trafficking and the formation of mDia2-dependent filopodia. Conversely, CLIC4 knockdown increased filopodium formation in an integrin-dependent manner, a phenotype rescued by wild-type CLIC4 but not by the trafficking-incompetent mutant CLIC4(C35A). Furthermore, CLIC4 accelerated LPA-induced filopodium retraction. We conclude that through profilin-1 binding, CLIC4 functions in a RhoA-mDia2-regulated signaling network to integrate cortical actin assembly and membrane protrusion. We propose that agonist-induced CLIC4 translocation provides a feedback mechanism that counteracts formin-driven filopodium formation.

New insights into the formation and the function of lamellipodia and ruffles in mesenchymal cell migration.

Lamellipodia and ruffles are veil-shaped cell protrusions composed of a highly branched actin filament meshwork assembled by the Arp2/3 complex. These structures not only hallmark the leading edge of cells adopting the adhesion-based mesenchymal mode of migration but are also thought to drive cell movement. Although regarded as textbook knowledge, the mechanism of formation of lamellipodia and ruffles has been revisited in the last years leveraging new technologies. Furthermore, recent observations have also challenged our current view of the function of lamellipodia and ruffles in mesenchymal cell migration. Here, I review this literature and compare it with older studies to highlight the controversies and the outstanding open issues in the field. Moreover, I outline simple and plausible explanations to reconcile conflicting results and conclusions. Finally, I integrate the mechanisms regulating actin-based protrusion in a unifying model that accounts for random and ballistic mesenchymal cell migration.

Knockout of the Arp2/3 complex in epidermis causes a psoriasis-like disease hallmarked by hyperactivation of transcription factor Nrf2.

The Arp2/3 complex assembles branched actin filaments, which are key to many cellular processes, but its organismal roles remain poorly understood. Here, we employed conditional Arpc4 knockout mice to study the function of the Arp2/3 complex in the epidermis. We found that depletion of the Arp2/3 complex by knockout of Arpc4 results in skin abnormalities at birth that evolve into a severe psoriasis-like disease hallmarked by hyperactivation of transcription factor Nrf2. Knockout of Arpc4 in cultured keratinocytes was sufficient to induce nuclear accumulation of Nrf2, upregulation of Nrf2 target genes and decreased filamentous actin levels. Furthermore, pharmacological inhibition of the Arp2/3 complex unmasked the role of branched actin filaments in Nrf2 regulation. Consistent with this, we revealed that Nrf2 associates with the actin cytoskeleton in cells and binds to filamentous actin in vitro Finally, we discovered that Arpc4 is downregulated in both human and mouse psoriatic epidermis. Thus, the Arp2/3 complex affects keratinocyte shape and transcriptome through an actin-based cell-autonomous mechanism that influences epidermal morphogenesis and homeostasis.

Flat clathrin lattices are dynamic actin-controlled hubs for clathrin-mediated endocytosis and signalling of specific receptors.

Clathrin lattices at the plasma membrane coat both invaginated and flat regions forming clathrin-coated pits and clathrin plaques, respectively. The function and regulation of clathrin-coated pits in endocytosis are well understood but clathrin plaques remain enigmatic nanodomains. Here we use super-resolution microscopy, molecular genetics and cell biology to show that clathrin plaques contain the machinery for clathrin-mediated endocytosis and cell adhesion, and associate with both clathrin-coated pits and filamentous actin. We also find that actin polymerization promoted by N-WASP through the Arp2/3 complex is crucial for the regulation of plaques but not pits. Clathrin plaques oppose cell migration and undergo actin- and N-WASP-dependent disassembly upon activation of LPA receptor 1, but not EGF receptor. Most importantly, plaque disassembly correlates with the endocytosis of LPA receptor 1 and down-modulation of AKT activity. Thus, clathrin plaques serve as dynamic actin-controlled hubs for clathrin-mediated endocytosis and signalling that exhibit receptor specificity.

New nuclear and perinuclear functions of formins.

Formin family proteins (formins) represent an evolutionary conserved protein family encoded in the genome of a wide range of eukaryotes. Formins are hallmarked by a formin homology 1 (FH1) domain juxtaposed to an FH2 domain whereby they control actin and microtubule dynamics. Not surprisingly, formins are best known as key regulators of the cytoskeleton in a variety of morphogenetic processes. However, mounting evidence implicates several formins in the assembly and organization of actin within and around the nucleus. In addition, actin-independent roles for formins have recently been discovered. In this mini-review, we summarize these findings and highlight the novel nuclear and perinulcear functions of formins. In light of the emerging new biology of formins, we also discuss the fundamental principles governing the versatile activity and multimodal regulation of these proteins.

Quantitative Proteomics Illuminates a Functional Interaction between mDia2 and the Proteasome.

Formin mDia2 is a cytoskeleton-regulatory protein that switches reversibly between a closed, autoinhibited and an open, active conformation. Although the open conformation of mDia2 induces actin assembly thereby controlling many cellular processes, mDia2 possesses also actin-independent and conformation-insensitive scaffolding roles related to microtubules and p53, respectively. Thus, we hypothesize that mDia2 may have other unappreciated functions and regulatory modes. Here we identify and validate proteasome and Ubiquitin as mDia2-interacting partners using stable isotope labeling with amino acids in cell culture-based quantitative proteomics and biochemistry, respectively. Although mDia2 is ubiquitinated, binds ubiquitinated proteins and free Ubiquitin, it is not a proteasome substrate. Surprisingly, knockdown of mDia2 increases the activity of the proteasome in vitro, whereas mDia2 overexpression has opposite effects only when it adopts the open conformation and cannot induce actin assembly. Consistently, a combination of candidate and unbiased proteome-wide analyses indicates that mDia2 regulates the cellular levels of proteasome substrate ß-catenin and a number of ubiquitinated actin-regulatory proteins. Hence, these findings add more complexity to the mDia2 activity cycle by showing that the open conformation may control actin dynamics also through actin-independent regulation of the proteasome.

PFA fixation enables artifact-free super-resolution imaging of the actin cytoskeleton and associated proteins.

Super-resolution microscopy (SRM) allows precise localization of proteins in cellular organelles and structures, including the actin cytoskeleton. Yet sample preparation protocols for SRM are rather anecdotal and still being optimized. Thus, SRM-based imaging of the actin cytoskeleton and associated proteins often remains challenging and poorly reproducible. Here, we show that proper paraformaldehyde (PFA)-based sample preparation preserves the architecture of the actin cytoskeleton almost as faithfully as gold-standard glutaraldehyde fixation. We show that this fixation is essential for proper immuno-based localization of actin-binding and actin-regulatory proteins involved in the formation of lamellipodia and ruffles, such as mDia1, WAVE2 and clathrin heavy chain, and provide detailed guidelines for the execution of our method. In summary, proper PFA-based sample preparation increases the multi-color possibilities and the reproducibility of SRM of the actin cytoskeleton and its associated proteins.

Invadosomes - shaping actin networks to follow mechanical cues.

Invadosomes are actin-based protrusions formed by cells in response to obstacles in their microenvironment, especially basement membranes and dense interstitial matrices. A versatile set of proteins controls assembly and dynamics of the actin networks at invadosomes and adhesive molecules link them with the extracellular matrix. Furthermore, polarized delivery of proteases makes invadosomes degradative. Therefore, invadosomes have been classically viewed as specialized protrusions involved in cell migration and remodeling of the microenvironment. Recent discoveries have considerably broadened this picture by showing that invadosomes respond to traction forces and can self-organize into dynamic arrays capable of following the topography of the substrate. Although these findings suggest that invadosomes may function as mechanosensors, this possibility has not been critically evaluated. In this review, we first summarize the organization and dynamics of actin in invadosomes and their superstructures with emphasis on force-production mechanisms. Next, we outline our current understanding of how mechanical cues impinge on invadosomes and modify their behavior. From this perspective, we provide an outlook of the outstanding open questions and the main challenges in the field.

Rapid Remodeling of Invadosomes by Gi-coupled Receptors: DISSECTING THE ROLE OF Rho GTPases.

Invadosomes are actin-rich membrane protrusions that degrade the extracellular matrix to drive tumor cell invasion. Key players in invadosome formation are c-Src and Rho family GTPases. Invadosomes can reassemble into circular rosette-like superstructures, but the underlying signaling mechanisms remain obscure. Here we show that Src-induced invadosomes in human melanoma cells (A375M and MDA-MB-435) undergo rapid remodeling into dynamic extracellular matrix-degrading rosettes by distinct G protein-coupled receptor agonists, notably lysophosphatidic acid (LPA; acting through the LPA1 receptor) and endothelin. Agonist-induced rosette formation is blocked by pertussis toxin, dependent on PI3K activity and accompanied by localized production of phosphatidylinositol 3,4,5-trisphosphate, whereas MAPK and Ca(2+) signaling are dispensable. Using FRET-based biosensors, we show that LPA and endothelin transiently activate Cdc42 through Gi, concurrent with a biphasic decrease in Rac activity and differential effects on RhoA. Cdc42 activity is essential for rosette formation, whereas G12/13-mediated RhoA-ROCK signaling suppresses the remodeling process. Our results reveal a Gi-mediated Cdc42 signaling axis by which G protein-coupled receptors trigger invadosome remodeling, the degree of which is dictated by the Cdc42-RhoA activity balance.

Initiation of lamellipodia and ruffles involves cooperation between mDia1 and the Arp2/3 complex.

Protrusion of lamellipodia and ruffles requires polymerization of branched actin filaments by the Arp2/3 complex. Although regulation of Arp2/3 complex activity has been extensively investigated, the mechanism of initiation of lamellipodia and ruffles remains poorly understood. Here, we show that mDia1 acts in concert with the Arp2/3 complex to promote initiation of lamellipodia and ruffles. We find that mDia1 is an epidermal growth factor (EGF)-regulated actin nucleator involved in membrane ruffling using a combination of knockdown and rescue experiments. At the molecular level, mDia1 polymerizes linear actin filaments, activating the Arp2/3 complex, and localizes within nascent and mature membrane ruffles. We employ functional complementation experiments and optogenetics to show that mDia1 cooperates with the Arp2/3 complex in initiating lamellipodia and ruffles. Finally, we show that genetic and pharmacological interference with this cooperation hampers ruffling and cell migration. Thus, we propose that the lamellipodium- and ruffle-initiating machinery consists of two actin nucleators that act sequentially to regulate membrane protrusion and cell migration.

SMIFH2 has effects on Formins and p53 that perturb the cell cytoskeleton.

Formin proteins are key regulators of the cytoskeleton involved in developmental and homeostatic programs, and human disease. For these reasons, small molecules interfering with Formins' activity have gained increasing attention. Among them, small molecule inhibitor of Formin Homology 2 domains (SMIFH2) is often used as a pharmacological Formin blocker. Although SMIFH2 inhibits actin polymerization by Formins and affects the actin cytoskeleton, its cellular mechanism of action and target specificity remain unclear. Here we show that SMIFH2 induces remodelling of actin filaments, microtubules and the Golgi complex as a result of its effects on Formins and p53. We found that SMIFH2 triggers alternated depolymerization-repolymerization cycles of actin and tubulin, increases cell migration, causes scattering of the Golgi complex, and also cytotoxicity at high dose. Moreover, SMIFH2 reduces expression and activity of p53 through a post-transcriptional, proteasome-independent mechanism that influences remodelling of the cytoskeleton. As the action of SMIFH2 may go beyond Formin inhibition, only short-term and low-dose SMIFH2 treatments minimize confounding effects induced by loss of p53 and cytotoxicity.