Advances and Challenges in Cell Biology for Cultured Meat.

Cultured meat is an emerging biotechnology that aims to produce meat from animal cell culture, rather than from the raising and slaughtering of livestock, on environmental and animal welfare grounds. The detailed understanding and accurate manipulation of cell biology are critical to the design of cultured meat bioprocesses. Recent years have seen significant interest in this field, with numerous scientific and commercial breakthroughs. Nevertheless, these technologies remain at a nascent stage, and myriad challenges remain, spanning the entire bioprocess. From a cell biological perspective, these include the identification of suitable starting cell types, tuning of proliferation and differentiation conditions, and optimization of cell-biomaterial interactions to create nutritious, enticing foods. Here, we discuss the key advances and outstanding challenges in cultured meat, with a particular focus on cell biology, and argue that solving the remaining bottlenecks in a cost-effective, scalable fashion will require coordinated, concerted scientific efforts. Success will also require solutions to nonscientific challenges, including regulatory approval, consumer acceptance, and market feasibility. However, if these can be overcome, cultured meat technologies can revolutionize our approach to food.

Microtubules in 3D cell motility.

Three-dimensional (3D) cell motility underlies essential processes, such as embryonic development, tissue repair and immune surveillance, and is involved in cancer progression. Although the cytoskeleton is a well-studied regulator of cell migration, most of what we know about its functions originates from studies conducted in two-dimensional (2D) cultures. This research established that the microtubule network mediates polarized trafficking and signaling that are crucial for cell shape and movement in 2D. In parallel, developments in light microscopy and 3D cell culture systems progressively allowed to investigate cytoskeletal functions in more physiologically relevant settings. Interestingly, several studies have demonstrated that microtubule involvement in cell morphogenesis and motility can differ in 2D and 3D environments. In this Commentary, we discuss these differences and their relevance for the understanding the role of microtubules in cell migration in vivo We also provide an overview of microtubule functions that were shown to control cell shape and motility in 3D matrices and discuss how they can be investigated further by using physiologically relevant models.

Mesenchymal Cell Invasion Requires Cooperative Regulation of Persistent Microtubule Growth by SLAIN2 and CLASP1.

Microtubules regulate signaling, trafficking, and cell mechanics, but the respective contribution of these functions to cell morphogenesis and migration in 3D matrices is unclear. Here, we report that the microtubule plus-end tracking protein (+TIP) SLAIN2, which suppresses catastrophes, is not required for 2D cell migration but is essential for mesenchymal cell invasion in 3D culture and in a mouse cancer model. We show that SLAIN2 inactivation does not affect Rho GTPase activity, trafficking, and focal adhesion formation. However, SLAIN2-dependent catastrophe inhibition determines microtubule resistance to compression and pseudopod elongation. Another +TIP, CLASP1, is also needed to form invasive pseudopods because it prevents catastrophes specifically at their tips. When microtubule growth persistence is reduced, inhibition of depolymerization is sufficient for pseudopod maintenance but not remodeling. We propose that catastrophe inhibition by SLAIN2 and CLASP1 supports mesenchymal cell shape in soft 3D matrices by enabling microtubules to perform a load-bearing function.

Molecular Pathway of Microtubule Organization at the Golgi Apparatus.

The Golgi apparatus controls the formation of non-centrosomal microtubule arrays important for Golgi organization, polarized transport, cell motility, and cell differentiation. Here, we show that CAMSAP2 stabilizes and attaches microtubule minus ends to the Golgi through a complex of AKAP450 and myomegalin. CLASPs stabilize CAMSAP2-decorated microtubules but are not required for their Golgi tethering. AKAP450 is also essential for Golgi microtubule nucleation, and myomegalin and CDK5RAP2 but not CAMSAP2 contribute to this function. In the absence of centrosomes, AKAP450- and CAMSAP2-dependent pathways of microtubule minus-end organization become dominant, and the presence of at least one of them is needed to maintain microtubule density. Strikingly, a compact Golgi can be assembled in the absence of both centrosomal and Golgi microtubules. However, CAMSAP2- and AKAP450-dependent Golgi microtubules facilitate Golgi reorientation and cell invasion in a 3D matrix. We propose that Golgi-anchored microtubules are important for polarized cell movement but not for coalescence of Golgi membranes.

Talin-KANK1 interaction controls the recruitment of cortical microtubule stabilizing complexes to focal adhesions.

The cross-talk between dynamic microtubules and integrin-based adhesions to the extracellular matrix plays a crucial role in cell polarity and migration. Microtubules regulate the turnover of adhesion sites, and, in turn, focal adhesions promote the cortical microtubule capture and stabilization in their vicinity, but the underlying mechanism is unknown. Here, we show that cortical microtubule stabilization sites containing CLASPs, KIF21A, LL5ß and liprins are recruited to focal adhesions by the adaptor protein KANK1, which directly interacts with the major adhesion component, talin. Structural studies showed that the conserved KN domain in KANK1 binds to the talin rod domain R7. Perturbation of this interaction, including a single point mutation in talin, which disrupts KANK1 binding but not the talin function in adhesion, abrogates the association of microtubule-stabilizing complexes with focal adhesions. We propose that the talin-KANK1 interaction links the two macromolecular assemblies that control cortical attachment of actin fibers and microtubules.

SCARECROW-LIKE23 and SCARECROW jointly specify endodermal cell fate but distinctly control SHORT-ROOT movement.

Intercellular signaling through trafficking of regulatory proteins is a widespread phenomenon in plants and can deliver positional information for the determination of cell fate. In the Arabidopsis root meristem, the cell fate determinant SHORT-ROOT (SHR), a GRAS domain transcription factor, acts as a signaling molecule from the stele to the adjacent layer to specify endodermal cell fate. Upon exiting the stele, SHR activates another GRAS domain transcription factor, SCARCROW (SCR), which, together with several BIRD/INDETERMINATE DOMAIN proteins, restricts movement of SHR to define a single cell layer of endodermis. Here we report that endodermal cell fate also requires the joint activity of both SCR and its closest homologue SCARECROW-LIKE23 (SCL23). We show that SCL23 protein moves with zonation-dependent directionality. Within the meristem, SCL23 exhibits short-ranged movement from ground tissue to vasculature. Away from the meristem, SCL23 displays long-range rootward movement into meristematic vasculature and a bidirectional radial spread, respectively. As a known target of SHR and SCR, SCL23 also interacts with SCR and SHR and can restrict intercellular outspread of SHR without relying on nuclear retention as SCR does. Collectively, our data show that SCL23 is a mobile protein that controls movement of SHR and acts redundantly with SCR to specify endodermal fate in the root meristem.

Arabidopsis BIRD Zinc Finger Proteins Jointly Stabilize Tissue Boundaries by Confining the Cell Fate Regulator SHORT-ROOT and Contributing to Fate Specification.

Plant cells cannot rearrange their positions; therefore, sharp tissue boundaries must be accurately programmed. Movement of the cell fate regulator SHORT-ROOT from the stele to the ground tissue has been associated with transferring positional information across tissue boundaries. The zinc finger BIRD protein JACKDAW has been shown to constrain SHORT-ROOT movement to a single layer, and other BIRD family proteins were postulated to counteract JACKDAW's role in restricting SHORT-ROOT action range. Here, we report that regulation of SHORT-ROOT movement requires additional BIRD proteins whose action is critical for the establishment and maintenance of the boundary between stele and ground tissue. We show that BIRD proteins act in concert and not in opposition. The exploitation of asymmetric redundancies allows the separation of two BIRD functions: constraining SHORT-ROOT spread through nuclear retention and transcriptional regulation of key downstream SHORT-ROOT targets, including SCARECROW and CYCLIND6. Our data indicate that BIRD proteins promote formative divisions and tissue specification in the Arabidopsis thaliana root meristem ground tissue by tethering and regulating transcriptional competence of SHORT-ROOT complexes. As a result, a tissue boundary is not "locked in" after initial patterning like in many animal systems, but possesses considerable developmental plasticity due to continuous reliance on mobile transcription factors.

Rb and FZR1/Cdh1 determine CDK4/6-cyclin D requirement in C. elegans and human cancer cells.

Cyclin-dependent kinases 4 and 6 (CDK4/6) in complex with D-type cyclins promote cell cycle entry. Most human cancers contain overactive CDK4/6-cyclin D, and CDK4/6-specific inhibitors are promising anti-cancer therapeutics. Here, we investigate the critical functions of CDK4/6-cyclin D kinases, starting from an unbiased screen in the nematode Caenorhabditis elegans. We found that simultaneous mutation of lin-35, a retinoblastoma (Rb)-related gene, and fzr-1, an orthologue to the APC/C co-activator Cdh1, completely eliminates the essential requirement of CDK4/6-cyclin D (CDK-4/CYD-1) in C. elegans. CDK-4/CYD-1 phosphorylates specific residues in the LIN-35 Rb spacer domain and FZR-1 amino terminus, resembling inactivating phosphorylations of the human proteins. In human breast cancer cells, simultaneous knockdown of Rb and FZR1 synergistically bypasses cell division arrest induced by the CDK4/6-specific inhibitor PD-0332991. Our data identify FZR1 as a candidate CDK4/6-cyclin D substrate and point to an APC/C(FZR1) activity as an important determinant in response to CDK4/6-inhibitors.

CFEOM1-associated kinesin KIF21A is a cortical microtubule growth inhibitor.

Mechanisms controlling microtubule dynamics at the cell cortex play a crucial role in cell morphogenesis and neuronal development. Here, we identified kinesin-4 KIF21A as an inhibitor of microtubule growth at the cell cortex. In vitro, KIF21A suppresses microtubule growth and inhibits catastrophes. In cells, KIF21A restricts microtubule growth and participates in organizing microtubule arrays at the cell edge. KIF21A is recruited to the cortex by KANK1, which coclusters with liprin-α1/ß1 and the components of the LL5ß-containing cortical microtubule attachment complexes. Mutations in KIF21A have been linked to congenital fibrosis of the extraocular muscles type 1 (CFEOM1), a dominant disorder associated with neurodevelopmental defects. CFEOM1-associated mutations relieve autoinhibition of the KIF21A motor, and this results in enhanced KIF21A accumulation in axonal growth cones, aberrant axon morphology, and reduced responsiveness to inhibitory cues. Our study provides mechanistic insight into cortical microtubule regulation and suggests that altered microtubule dynamics contribute to CFEOM1 pathogenesis.

PLA2R1 kills cancer cells by inducing mitochondrial stress.

Little is known about the biological functions of the phospholipase A2 receptor (PLA2R1) except that it has the ability to bind a few secreted phospholipases A2 (sPLA2's). We have previously shown that PLA2R1 regulates senescence in normal human cells. In this study, we investigated the ability of PLA2R1 to control cancer cell growth. Analysis of expression in cancer cells indicates a marked PLA2R1 decrease in breast cancer cell lines compared to normal or nontransformed human mammary epithelial cells. Accordingly, PLA2R1 ectopic expression in PLA2R1-negative breast cancer cell lines led to apoptosis, whereas a prosenescence response was predominantly triggered in normal cells. PLA2R1 structure-function studies and the use of chemical inhibitors of sPLA2-related signaling pathways suggest that the effect of PLA2R1 is sPLA2-independent. Functional experiments demonstrate that PLA2R1 regulation of cell death is driven by a reactive oxygen species (ROS)-dependent mechanism. While screening for ROS-producing complexes involved in PLA2R1 biological responses, we identified a critical role for the mitochondrial electron transport chain in PLA2R1-induced ROS production and cell death. Taken together, this set of data provides evidence for an important role of PLA2R1 in controlling cancer cell death by influencing mitochondrial biology.

Microtubule plus-end tracking proteins SLAIN1/2 and ch-TOG promote axonal development.

Development, polarization, structural integrity, and plasticity of neuronal cells critically depend on the microtubule network and its dynamic properties. SLAIN1 and SLAIN2 are microtubule plus-end tracking proteins that have been recently identified as regulators of microtubule dynamics. SLAINs are targeted to microtubule tips through an interaction with the core components of microtubule plus-end tracking protein network, End Binding family members. SLAINs promote persistent microtubule growth by recruiting the microtubule polymerase ch-TOG to microtubule plus-ends. Here, we show that SLAIN1/2 and ch-TOG-proteins are highly enriched in brain and are expressed throughout mouse brain development. Disruption of the SLAIN-ch-TOG complex in cultured primary rat hippocampal neurons by RNA interference-mediated knockdown and a dominant-negative approach perturbs microtubule growth by increasing catastrophe frequency and inhibits axon extension during neuronal development. Our study shows that proper control of microtubule dynamics is important for axon elongation in developing neurons.

Induction of EMT by twist proteins as a collateral effect of tumor-promoting inactivation of premature senescence.

Twist1 and Twist2 are major regulators of embryogenesis. Twist1 has been shown to favor the metastatic dissemination of cancer cells through its ability to induce an epithelial-mesenchymal transition (EMT). Here, we show that a large fraction of human cancers overexpress Twist1 and/or Twist2. Both proteins override oncogene-induced premature senescence by abrogating key regulators of the p53- and Rb-dependent pathways. Twist1 and Twist2 cooperate with Ras to transform mouse embryonic fibroblasts. Interestingly, in epithelial cells, the oncogenic cooperation between Twist proteins and activated mitogenic oncoproteins, such as Ras or ErbB2, leads to complete EMT. These findings suggest an unanticipated direct link between early escape from failsafe programs and the acquisition of invasive features by cancer cells.

Weekly administration of paclitaxel induces long-term aneugenicity in nude mice.

We investigated the potential in vivo aneugenic effects associated with paclitaxel treatment. For this purpose, we treated female nude mice with paclitaxel using doses equivalent to those used in weekly schedules at the clinical level (three cycles of 30 mg/kg/week for three consecutive weeks followed by one resting week). We then evaluated the frequencies of micronucleated erythrocytes (MNE) in peripheral blood using the acridine orange micronucleus assay. The frequency of MNE was evaluated after 24 h and 168 h of administration of the last dose of each paclitaxel cycle (STA mice group) as well as after one year of the first dose of treatment (LTA mice group). We also analyzed the cytology of peripheral blood and bone marrows obtained from these mice at each time period. In the STA mice group, three cycles of paclitaxel induced a 2.4-fold increase in MNE frequencies compared to the control group (p < 0.01). This effect was observed after 24 h of the last dose of each chemotherapy cycle and persisted at least for 168 h. In the LTA mice group, paclitaxel-treated mice presented a 1.8-fold increase in the MNE frequency (p = 0.01) indicating that paclitaxel-induced MNE increase lasted for at least one year. Although the appearance of micronuclei in erythrocytes and granulocytes in peripheral blood and bone marrow cytological smears, there was no evidence of myeloproliferative disease. The present data therefore indicate an aneugenic potential of paclitaxel for humans, which should be considered in the risk-benefit analysis of its increasing clinical use.