Coping with loss: cell adaptation to cytoskeleton disruption

Unravelling the role of cytoskeleton regulators may be complicated by adaptations to experimental manipulations. In this issue of Developmental Cell, Cerikan et al. (2016) reveal how acute effects of DOCK6 RhoGEF depletion on RAC1 and CDC42 activation are reversed over time by compensatory mechanisms that re-establish cellular homeostasis

Sensor potency of the moonlighting enzyme-decorated cytoskeleton

Background: There is extensive evidence for the interaction of metabolic enzymes with the eukaryotic cytoskeleton. The significance of these interactions is far from clear. Presentation of the hypothesis: In the cytoskeletal integrative sensor hypothesis presented here, the cytoskeleton senses and integrates the general metabolic activity of the cell. This activity depends on the binding to the cytoskeleton of enzymes and, depending on the nature of the enzyme, this binding may occur if the enzyme is either active or inactive but not both. This enzyme-binding is further proposed to stabilize microtubules and microfilaments and to alter rates of GTP and ATP hydrolysis and their levels. Testing the hypothesis: Evidence consistent with the cytoskeletal integrative sensor hypothesis is presented in the case of glycolysis. Several testable predictions are made. There should be a relationship between post-translational modifications of tubulin and of actin and their interaction with metabolic enzymes. Different conditions of cytoskeletal dynamics and enzyme-cytoskeleton binding should reveal significant differences in local and perhaps global levels and ratios of ATP and GTP. The different functions of moonlighting enzymes should depend on cytoskeletal binding. Implications of the hypothesis: The physical and chemical effects arising from metabolic sensing by the cytoskeleton would have major consequences on cell shape, dynamics and cell cycle progression. The hypothesis provides a framework that helps the significance of the enzyme-decorated cytoskeleton be determined

Interaction of microtubules and actin during the post-fusion phase of exocytosis

Exocytosis is the intracellular trafficking step where a secretory vesicle fuses with the plasma membrane to release vesicle content. Actin and microtubules both play a role in exocytosis; however, their interplay is not understood. Here we study the interaction of actin and microtubules during exocytosis in lung alveolar type II (ATII) cells that secrete surfactant from large secretory vesicles. Surfactant extrusion is facilitated by an actin coat that forms on the vesicle shortly after fusion pore opening. Actin coat compression allows hydrophobic surfactant to be released from the vesicle. We show that microtubules are localized close to actin coats and stay close to the coats during their compression. Inhibition of microtubule polymerization by colchicine and nocodazole affected the kinetics of actin coat formation and the extent of actin polymerisation on fused vesicles. In addition, microtubule and actin cross-linking protein IQGAP1 localized to fused secretory vesicles and IQGAP1 silencing influenced actin polymerisation after vesicle fusion. This study demonstrates that microtubules can influence actin coat formation and actin polymerization on secretory vesicles during exocytosis

The Antithetic Roles of IQGAP2 and IQGAP3 in Cancers

Simple Summary The IQ motif-containing GTPase-activating protein family is comprised of three signal scaffolding proteins that regulate a variety of biological functions by aiding signal transduction in cells. IQGAPs induce numerous cancer-related processes, including proliferation, apoptosis, migration, invasion, and angiogenesis. In comparison to IQGAP1, IQGAP2 and IQGAP3 were less researched. In this review, we comprehensively reviewed the significant roles of IQGAP2 and IQGAP3 in cancer-associated pathways as well as the role in carcinogenesis and progression of different cancer entities. Abstract The scaffold protein family of IQ motif-containing GTPase-activating proteins (IQGAP1, 2, and 3) share a high degree of homology and comprise six functional domains. IQGAPs bind and regulate the cytoskeleton, interact with MAP kinases and calmodulin, and have GTPase-related activity, as well as a RasGAP domain. Thus, IQGAPs regulate multiple cellular processes and pathways, affecting cell division, growth, cell–cell interactions, migration, and invasion. In the past decade, significant evidence on the function of IQGAPs in signal transduction during carcinogenesis has emerged. Compared with IQGAP1, IQGAP2 and IQGAP3 were less analyzed. In this review, we summarize the different signaling pathways affected by IQGAP2 and IQGAP3, and the antithetic roles of IQGAP2 and IQGAP3 in different types of cancer. IQGAP2 expression is reduced and plays a tumor suppressor role in most solid cancer types, while IQGAP3 is overexpressed and acts as an oncogene. In lymphoma, for example, IQGAPs have partially opposite functions. There is considerable evidence that IQGAPs regulate a multitude of pathways to modulate cancer processes and chemoresistance, but some questions, such as how they trigger this signaling, through which domains, and why they play opposite roles on the same pathways, are still unanswered

Regulation of the Actin Cytoskeleton via Rho GTPase Signalling in Dictyostelium and Mammalian Cells: A Parallel Slalom

Both Dictyostelium amoebae and mammalian cells are endowed with an elaborate actin cytoskeleton that enables them to perform a multitude of tasks essential for survival. Although these organisms diverged more than a billion years ago, their cells share the capability of chemotactic migration, large- scale endocytosis, binary division effected by actomyosin contraction, and various types of adhesions to other cells and to the extracellular environment. The composition and dynamics of the transient actin-based structures that are engaged in these processes are also astonishingly similar in these evolutionary distant organisms. The question arises whether this remarkable resemblance in the cellular motility hardware is accompanied by a similar correspondence in matching software, the signalling networks that govern the assembly of the actin cytoskeleton. Small GTPases from the Rho family play pivotal roles in the control of the actin cytoskeleton dynamics. Indicatively, Dictyostelium matches mammals in the number of these proteins. We give an overview of the Rho signalling pathways that regulate the actin dynamics in Dictyostelium and compare them with similar signalling networks in mammals. We also provide a phylogeny of Rho GTPases in Amoebozoa, which shows a variability of the Rho inventories across different clades found also in Metazoa

Neuronal functions of RIM3γ and RIM4γ

The large isoforms of the Rab3 interacting molecule (RIM) family, RIM1α/β and RIM2α/β are integral components of the cytomatrix of the presynaptic active zone. Through multiple interactions with other active zone proteins they are involved in regulating several steps of presynaptic neurotransmitter release. The RIM protein family contains two additional isoforms, RIM3γ and RIM4γ, whose functions remain to be elucidated. In this study we could show that RIM3γ and RIM4γ are key regulators in neuronal growth involved in the establishment of axons and dendrites, formation of synapses and dendritic spines. Furthermore, the loss of RIM3γ leads to a dispersion of the Golgi apparatus whereas the loss of RIM4γ induces the condensation of the Golgi in the cell's soma. These findings provide first hints that the small γ-Rims are involved in neuronal growth by regulating processes of vesicular traffic. Live cell imaging of Rab-protein marked vesicles in RIM3γ and RIM4γ knock-down neurons revealed changes in the velocity and the path length of Rab11 and Rab8 vesicles, confirming that the loss RIM3γ and RIM4γ has an effect on intracellular transport routes. In an affinity purification mass spectrometry based search for novel γ-RIM interaction partners we identified a large set of proteins that have been associated with neuronal growth through the remodeling of the cytoskeleton or vesicular traffic and in addition a smaller group of synaptic proteins. A comparison of the individual affinity purification mass spectrometry approaches performed in different tissue fractions revealed that a cluster of new γ-RIM interaction partners was repeatedly detected. This cluster contained proteins involved in the remodeling of the cytoskeleton, site directed transport of vesicles and proteins that belong to signaling cascades important for axonal and dendritic growth. We performed first validation experiments using in vitro binding assays to decipherer the direct interactions of this complex. These experiments confirmed in vitro an interaction between γ-RIMs and the cytoskeleton regulator IQGAP3, the adhesion molecule plakophilin4 and Syd-1 an important initiator of synapse assembly. To get further insights in the cellular functions of RIM3γ and RIM4γ we generated constitutive knock-out mouse lines for both proteins. RIM3γ and RIM4γ knock-out mice are viable and not distinguishable from wild type litters until 20 days after birth. Around an age of three weeks RIM4γ knock-out mice develop a strong episodic motor phenotype, most prominent in the hind limbs, whereas RIM3γ knock-out mice show no obvious phenotype. Behavioral tests of motor coordination revealed that RIM4γ knock-out mice suffer also during phenotypic inconspicuous periods from milder coordinative disturbances suggesting impairments in cerebellar functions. Interestingly, after the induction of neuronal activity in an animal model of epilepsy RIM4γ transcripts were strongly upregulated in the hippocampus 12 hours after status epilepticus whereas RIM3γ transcripts were downregulated in the hippocampal subregion dentate gyrus after 36 hours. These findings suggest that the γ-RIMs might be either involved in pathological changes occurring during epileptogenesis or in neuroprotective mechanisms in response to increased network activity. Taken together, the results of this study provide new insights into the function of RIM3γ and RIM4γ in the development and the function of a healthy brain and form the basis for future studies on the precise understanding of the role of RIM3γ and RIM4γ during the pathogenesis of neurological disorders

DYNAMIC INTERPLAY BETWEEN CLEAVAGE FURROW PROTEINS IN CELLULAR MECHANORESPONSIVENESS

Cell shape changes associated with processes like cytokinesis and motility proceed on several second time-scales. However, they are derived from much faster molecular events occurring much faster, including protein-protein interactions, filament assembly, and force generation. How these fast molecular dynamics define cellular outcomes remain unknown. While accumulation of cytoskeletal elements during shape change is often driven by signaling pathways, mechanical stresses also direct proteins. A myosin II-based mechanosensory system controls cellular contractility and shape during cytokinesis and under applied stress. In Dictyostelium, this system tunes myosin II accumulation under mechanical stress by feedback through the actin network, particularly through the crosslinker cortexillin I. Cortexillin-binding IQGAP proteins are major regulators of this system. We examined the dynamic interplay between these key cytoskeletal proteins using fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS), defining the short time-scale dynamics of these players during cytokinesis and under mechanical stress. Actin and its polar cortex-enriched crosslinkers showed sub-second recovery, while equatorially enriched proteins including cortexillin I, IQGAP2, and myosin II recovered in 1-5 seconds. Mobility of these equatorial proteins was greatly reduced at the furrow, compared to their interphase dynamics. This mobility shift did not arise from a single biochemical event, but rather from global inhibition of protein dynamics by mechanical stress-associated changes in cytoskeletal structure. Thus, the equatorial proteins are stabilized under mechanical stress, which likely enables them to generate contractility at the furrow. We further expanded our genetic and biochemical understanding of this mechanosensory system using a proteomics approach to identify relevant protein-protein interactions. We identified that, in addition to binding to each other, both cortexillin I and IQGAP2 also interact with myosin II under conditions that prevent myosin II-F-actin binding. Thus, cooperativity between This validates the high crosstalk occurring between various mechanosensitive elements through macromolecular assemblies may provide a new mechanism for regulating cellular contractility. Mechanical tuning of contractile protein dynamics provides robustness to the cytoskeletal framework responsible for regulating cell shape and contributes to the fidelity of cytokinesis