Using Optogenetics to Investigate the Shared Mechanisms of Apical-Basal Polarity and Mitosis

The initiation of apical-basal (AB) polarity and the process of mitotic cell division are both characterised by the generation of specialised plasma membrane and cortical domains. These are generated using shared mechanisms, such as asymmetric protein accumulation, Rho GTPase signalling, cytoskeletal reorganisation, vesicle trafficking, and asymmetric phosphoinositide distribution. In epithelial tissue, the coordination of AB polarity and mitosis in space and time is important both during initial epithelial development and to maintain tissue integrity and ensure appropriate cell differentiation at later stages. Whilst significant progress has been made in understanding the mechanisms underlying cell division and AB polarity, it has so far been challenging to fully unpick the complex interrelationship between polarity, signalling, morphogenesis, and cell division. However, the recent emergence of optogenetic protein localisation techniques is now allowing researchers to reversibly control protein activation, localisation, and signalling with high spatiotemporal resolution. This has the potential to revolutionise our understanding of how subcellular processes such as AB polarity are integrated with cell behaviours such as mitosis and how these processes impact whole tissue morphogenesis. So far, these techniques have been used to investigate processes such as cleavage furrow ingression, mitotic spindle positioning, and in vivo epithelial morphogenesis. This review describes some of the key shared mechanisms of cell division and AB polarity establishment, how they are coordinated during development and how the advance of optogenetic techniques is furthering this research field.


Introduction
Rhodopsin-based optogenetic approaches have revolutionised the field of neuroscience, allowing researchers to dissect the connectivity of neural circuits via the precise and rapid activation and deactivation of specific neurons [Deisseroth, 2015].More recently, many non-rhodopsin optogenetic systems have arisen, enabling a wide range of light-dependent techniques.For example, inducible gene expression [de Mena et al., 2018], manipulation of protein signalling [Tischer and Weiner, 2014;Farahani et al., 2021], and loss or gain of protein function [Guglielmi et al., 2016] can now be achieved with subcellular spatial resolution and a temporal resolution of milliseconds-seconds.
One of the areas of research to which optogenetics is particularly suited is to further understand how changes at the molecular scale affect cell behaviour and ultimately, tissue-scale changes.For example, the coordination of apical-basal (AB) polarity and mitotic cell division during development facilitates polarity establishment during development of epithelia and regulates epithelial tissue cohesion and cell behaviour in already established epithelia.However, it is still not fully understood how these two processes are integrated at the molecular level.AB polarity and mitosis share several common mechanisms that generate specialised domains within the cell cortex and plasma membrane, all of which can now be manipulated using optogenetics to further understand these processes.These include (a) Rho GTPase signalling and cytoskeletal reorganisation, (b) vesicular trafficking, and (c) changes to membrane phosphoinositide composition (Fig. 1).These mechanisms deliver and reinforce the asymmetric distribution of polarity proteins, and this is coordinated (iii) Correct orientation and localisation of the spindle and actomyosin contractile ring is important for midbody and AB polarity placement in polarised epithelial cysts.b Vesicular and protein transport.Vesicles are trafficked to the midbody during cytokinesis (direction of trafficking indicated by arrows).This is required for abscission, and delivery of apical proteins for AMIS (orange) formation in AB polarising epithelial cells.c PIP distribution.PIPs are asymmetrically distributed in dividing cells and AB polarised epithelial cysts.In dividing cells, PI(4,5)P 2 is enriched at the cleavage furrow and PI(3,4,5) P 3 found at the poles.In epithelial cysts, PI(3,4)P 2 and PI(4,5)P 2 are enriched apically, whilst PI(3,4,5)P 3 (and to a lesser extent PI(4,5)P 2 ) is found basolaterally.

Phosphatidylinositol phosphate distribution
PI(4,5)P2 cleavage furrow accumulation AB polarised PIP distribution during cell mitosis to enable cytokinesis and to correctly place the apical, junctional, and basolateral domains in daughter cells.This review summarises our current understanding of how AB polarity establishment and cell mitosis are coordinated during development.It then explores which optogenetic methods can be best used to further our understanding of these processes, in particular how they operate in the context of tissue morphogenesis during development.

De Novo AB Polarisation of Hollowing Epithelial Tubes/Cavities
A key example where AB polarity and mitosis must be coordinated is de novo AB polarity establishment within the centre of hollowing epithelial tubes and cavities.During this process, the localisation of apical surfaces is determined by the apical membrane initiation site (AMIS), which is aligned with the mitotic midbody of dividing epithelial progenitor cells.The AMIS is a transient structure comprising scaffolding and junctional proteins that marks the point on the cell membrane where apical proteins will be deposited [Bryant et al., 2010;Blasky et al., 2015;Buckley and St Johnston, 2022].During epithelial progenitor cell mitoses, it is now well established that the mitotic midbody is sufficient for directing the localisation of the AMIS, therefore, determining both where cytokinesis will occur and where lumens will initiate [Schlüter et al., 2009;Li et al., 2014b;Wang et al., 2014;Klinkert et al., 2016;Mangan et al., 2016;Rathbun et al., 2020].After initial AB polarisation and epithelial establishment, subsequent cell divisions also position the midbody towards the apical surface [Jaffe et al., 2008;Wang et al., 2014;Bai et al., 2020], where they persist long after cell division [Luján et al., 2016].Therefore, defects in midbody position (for example, by misorientation of the mitotic spindle, ectopic cleavage furrow localisation, or other cell division defect) results in multiple or fragmented lumens or apical domains due to the mislocalisation of the AMIS [Ciruna et al., 2006;Tawk et al., 2007;Jaffe et al., 2008;Hao et al., 2010;Quesada-Hernández et al., 2010;Rodriguez-Fraticelli et al., 2010;Durgan et al., 2011;Žigman et al., 2011, 2014;Buckley et al., 2013;Luján et al., 2016;Gao et al., 2017].Regulation of mitosis location and timing and persistence of an apical midbody is therefore likely a common mechanism to coordinate the formation of the apical and junctional domains within a dynamically growing tissue and its dysregulation has been implicated in epithelial tumorigenesis [Luján et al., 2016;Jewett and Prekeris, 2018].Despite the clear importance of the mitotic midbody in directing apical membrane localisation via AMIS positioning, recent work has demonstrated that AMIS localisation initially occurs upstream and independently of cell division, via cadherin-mediated cellcell adhesions [Buckley et al., 2013;Liang et al., 2022].This suggests that the role of the midbody is to align cell division with the already forming apical membrane, therefore building an organised epithelium [Buckley and St Johnston, 2022;Liang et al., 2022].
Several outstanding questions remain regarding how AB polarity and cell division are aligned during hollowing epithelial lumenogenesis.While spindle orientation and midbody positioning determines the location of the apical surface, apically associated proteins are themselves important in controlling spindle orientation.For example, the apically associated Rho GTPase Cdc42 plays an important role in regulating spindle orientation [Jaffe et al., 2008;Kieserman and Wallingford, 2009;Qin et al., 2010;Rodriguez-Fraticelli et al., 2010].Knockdown of Cdc42 or its guanine nucleotide exchange factors (GEFs) including Tuba and Intersectin 2 (ITSN2) causes multi-lumen phenotypes [Martin-Belmonte et al., 2007;Jaffe et al., 2008;Qin et al., 2010;Rodriguez-Fraticelli et al., 2010].This raises the question of what the hierarchy of AB polarity establishment and cell mitosis location and orientation within a developing epithelium is.The scaffolding protein Partitioning defective 3 homolog (PAR-3) has been implicated in centrosome localisation [Hong et al., 2010;Jiang et al., 2015], so it is possible that the early localisation of AMIS-associated proteins prior to cell division directs centrosome (and therefore mitotic spindle) positioning [Buckley et al., 2013;Buckley and Clarke, 2014].Once mitosis is underway, key junctional and scaffolding proteins become concentrated in the cleavage furrow [Tawk et al., 2007;Schlüter et al., 2009;Buckley et al., 2013;Li et al., 2014b;Symonds et al., 2020], but the mechanisms by which this occurs is unknown.Lastly, while there have been several recent advances in our understanding of how proteins required for both AB polarity and mitosis are trafficked to the mitotic midbody (discussed below), there is still not a consensus over which are the key proteins responsible and what is their hierarchy of action.
Optogenetic approaches are well placed to start to answer these questions since they provide a method to directly perturb specific nodes of the complex and interrelated mechanisms of adhesion, AB polarity, and cell division.

Rho GTPase Signalling and Cytoskeletal Organisation
Rho family GTPases include proteins such as RhoA, Rac1, and Cdc42 that play important regulatory roles in processes such as cell polarity, cell migration, cell adhesion, and cell cycle progression [Mack and Georgiou, 2014].Rho GTPases switch between inactive GDP-bound to active GTP-bound forms, regulated by activating guanine nucleotide exchange factors (GEFs) and deactivating GTPase-activating proteins (GAPs).One of their major downstream effects is actomyosin cytoskeleton reorganisation.For example, RhoA, Rac1, and Cdc42 activation can trigger actin polymerisation through recruitment of actin nucleating diaphanous-related formin proteins.Non-muscle myosin-II (NMYII) contractility is regulated by RhoA via its downstream effector Rho kinase (ROCK) and by Cdc42 via myotonic-dystrophy-kinaserelated Cdc42-binding kinase (MRCK).ROCK and MRCK also regulate actin tethering to the plasma membrane by phosphorylation of ezrin-radixin-moesin proteins [Sit and Manser, 2011].
During mitotic cell division, uniformity in cortical tension in cells during metaphase is broken by spatial regulation of RhoA that leads to the assembly of the actomyosin contractile ring at the cell equator and relaxation of contractility at the cell poles [Taubenberger et al., 2020].The RhoGEF epithelial cell transforming sequence 2 (Ect2) is recruited by the centralspindlin complex at the spindle midzone and accumulates at the overlying equatorial plasma membrane [Yüce et al., 2005;Nishimura and Yonemura, 2006;Su et al., 2011].This restricts RhoA activity to the equator, leading to the formation of the actomyosin contractile ring during anaphase (Fig. 1aii), which allows cleavage furrow ingression during anaphase, and intercellular bridge and midbody formation during telophase [Ramkumar and Baum, 2016].
There are strong links between actomyosin contractility and polarity establishment in early mouse and Caenorhabditis elegans embryos [Munro et al., 2004;Schonegg and Hyman, 2006;Goehring et al., 2011;Zhu et al., 2017] and other cell types such as the Drosophila neuroblast [Oon and Prehoda, 2021].The involvement of RhoA and actomyosin contractility in epithelial AB polarity establishment is still unclear, but recent evidence suggests it might play a role in apical polarisation.During the de novo polarisation of the mouse epiblast, active NMYII (indicated by phosphorylated myosin light chain II) localises in the centre of the tissue, coincident with the forming apical surface, while ECM-associated protein, β1 integrin, localises on the outer basal surface of the tissue [Molè et al., 2021].Disruption of β1 integrin signalling causes the ectopic accumulation of both phosphorylated myosin light chain II and apical proteins such as partitioning defective 6 homolog (PAR-6) on the basal surface, leading to tissue disruption [Molè et al., 2021].Inversion of AB polarity is also seen following disruption of β1 integrin or its ligand laminin in many other models of de novo polarising epithelial tubes and cysts; both in vitro (for example, Madin-Darby canine kidney (MDCK) cells, primary luminal mammary epithelial cells, mouse embryo stem cells [O'Brien et al., 2001;Yu et al., 2005Yu et al., , 2008;;Akhtar and Streuli, 2012;Bedzhov and Zernicka-Goetz, 2014]) and in vivo (for example, zebrafish neural tube, C. elegans intestine [Rasmussen et al., 2012;Buckley et al., 2013]).These effects are thought to be mediated by ectopic basal RhoA-ROCK signalling, which is usually inhibited by β1 integrin signalling [Bryant et al., 2014], since RhoA or ROCK inhibition can rescue these phenotypes [Yu et al., 2008;Molè et al., 2021].
RhoA signalling is also important for the maintenance of the actomyosin belt at the adherens junctions of epithelia [Smutny et al., 2010;Priya et al., 2013].RhoA activation at the cell-cell junctions recruits and activates NMYIIA, increasing junctional tension and regulating adherens and tight junction localisation [Terry et al., 2011;Ratheesh et al., 2012;Reyes et al., 2014].Interestingly, the cytokinetic proteins Anillin, Centralspindlin and Ect2 that are associated with the actomyosin contractile ring also recruit and activate RhoA at the cell-cell junctions of epithelial cells during interphase [Ratheesh et al., 2012;Reyes et al., 2014;Budnar et al., 2019].
As already discussed, the apically associated Rho GTPase Cdc42 is important for both apical domain formation and regulation of mitotic spindle orientation and the hierarchy of these events is unknown.The latter may depend upon downstream aPKC-mediated phosphorylation of leucine/glycine/asparagine-repeat-containing protein (LGN) to exclude the NuMA/LGN/Gαi spindle orientation complex from the apical cortex, orienting the spindle perpendicular to the AB axis [Hao et al., 2010;Zheng et al., 2010;Durgan et al., 2011].

Optogenetic Manipulation of Rho GTPases and Cytoskeletal Organisation
There is a widespread interest in optogenetic techniques that modulate cytoskeletal organisation and several optogenetic systems have recently emerged to study Cells Tissues Organs 2024;213:161-179 DOI: 10.1159/000528796 a range of cell biological processes such as cleavage furrow formation, spindle orientation, front-rear polarity establishment, and epithelial morphogenesis.A popular approach is to modulate Rho GTPase activity by localising a relevant GEF or GAP to a specific region of the cell membrane (for example, Fig. 2a).Typically, a minimal protein is recruited, such as the catalytic Dbl-homology (DH) and regulatory pleckstrin-homology (PH) domains of GEFs [Rossman et al., 2005].This allows endogenous signalling activation/deactivation without overexpression of the full-length protein, therefore reducing overexpression phenotypes.Such techniques have the potential to help answer outstanding questions such as what role actomyosin contractility plays in apical membrane localisation and to unravel how AB polarity establishment and mitosis location and orientation interrelate.
Optogenetic manipulation of GTPases such as RhoA or Rho1 can be used to control actomyosin contractility by downstream activation of NMYII.For example, using the blue-light-specific LOV-domain-based optogenetic protein interaction system tunable light-inducible dimerisation tags, TULIPs [Strickland et al., 2012], recruitment of the catalytic DH domain of the RhoA-specific GEF, Leukaemia-associated Rho guanine nucleotide exchange factor (LARG), to a subcellular region of the plasma membrane of HeLa cells was sufficient to induce ectopic "cleavage furrows" in non-adherent cells during interphase (Fig. 2b) [Wagner and Glotzer, 2016].In the macrophage-like RAW 264.7 cell line, recruitment of the DH/ PH domain of LARG to the plasma membrane via the LOV-domain-based improved light-inducible dimer (iLID) heterodimerisation system [Guntas et al., 2015], showed that RhoA-mediated cleavage furrow formation occurs via actomyosin contraction and directed plasma membrane flow that decreases membrane tension and increases endocytosis at the cleavage furrow [Castillo-Badillo et al., 2020].
Optogenetic RhoA or Rho1 manipulation in vivo is now allowing advances in understanding of how NMYII contractility at a subcellular scale drives epithelial morphogenesis at the tissue level.For example, during Drosophila gastrulation, a ventral furrow is formed (partly via NMYII-mediated apical constriction of the ventral epithelial surface), while the dorsal surface does not invaginate (Fig. 2c).Two optogenetic studies from the De Renzis laboratory used the blue-light-mediated cryptochrome protein heterodimerisation system (CRY2/CIBN), to target the catalytic domain of RhoGEF2 (and therefore NMYII contractility) to ectopic membrane regions of the Drosophila blastoderm.These studies demonstrated first; that ectopic Rho1 activation at the apical membrane of the dorsal epithelium was sufficient to induce tissue invagination (Fig. 2ci) [Izquierdo et al., 2018], and second; that ectopic Rho1 activation at the basal side of the ventral epithelium prevented apical invagination (Fig. 2cii) [Krueger et al., 2018].These experiments suggest that both apical constriction and basal relaxation are needed for successful tissue folding.To achieve the subcellular specificity needed for these experiments, a combination of two-photon illumination and basal-specific anchor proteins was used.Optogenetic control of actomyosin has since been used to further explore the role of NMYII contractility in morphogenetic processes such as tissue folding, nuclear positioning and cellularisation in the in vivo Drosophila embryo [Deneke et al., 2019;Krueger et al., 2019bKrueger et al., , 2020;;Bhide et al., 2021].
Optogenetic approaches have also been used to compare the effects of activation and inhibition of NMYII contractility.For example, the CRY2/CIBN system was used within MDCK monolayers to achieve subcellular modulation of RhoA signalling by recruiting the catalytic DH/PH domain of its GEF ARHGEF11 to either the cell membrane (activating RhoA) or to mitochondria (sequestering the GEF and therefore inhibiting RhoA at the cell membrane) [Valon et al., 2017].This resulted in changes to cellular contractility and to downstream Yes-associated protein (YAP) mechanosensory signalling [Valon et al., 2017].These same "optoGEF-RhoA" constructs were used to manipulate RhoA activity at the rear or front of Xenopus cranial neural crest cell clusters in vivo and ex vivo, demonstrating that high rear and low front contractility is necessary and sufficient for collective chemotaxis [Shellard et al., 2018].Cryptochrome optogenetics has also been used to localise full-length Rho1 GEF or GAP to the apical membrane of the Drosophila germband epithelium, with differential effects on the apical-medial and junctional myosin pools [Herrera-Perez et al., 2021].
Relaxation of myosin contractility can also be optogenetically controlled independently of RhoA (and therefore of its other downstream effects), via the OptoMYPT system [Yamamoto et al., 2021].This uses the iLID optogenetic heterodimerisation system to recruit the protein phosphatase 1c binding domain of the myosin light chain phosphatase regulatory subunit, MYPT1, to the plasma membrane, resulting in dephosphorylation and inactivation of NMYII.Reduction in actomyosin contractility at both poles of dividing MDCK cells accelerated cleavage furrow ingression [Yamamoto et al., 2021], supporting the polar relaxation model of cleavage furrow formation [Verma et al., 2019].DOI: 10.1159/000528796 Manipulating mitotic spindle orientation and positioning presents another method to alter cytoskeletal organisation.Mitotic spindle orientation can be controlled optogenetically in mammalian cells and C. elegans em-bryos by engineering an interaction between NuMA and the cell cortex (Fig. 3a), bypassing Gαi and LGN [Fielmich et al., 2018;Okumura et al., 2018].Sustained optogenetic activation on one side of the cell caused spindle misplace- ment, generating asymmetric daughter cells, whereas moving the region of activation caused spindle rotation (Fig. 3b) [Okumura et al., 2018].Spindle rotation also occurs following optogenetic RhoA activation at both poles [Kelkar et al., 2022].Optogenetically induced regions of high actomyosin accumulation reduced pulling forces on the astral microtubules which, due to the broader distribution of NuMA, created unstable pulling forces that rotated the spindle.
While optogenetic manipulation of Cdc42 has not yet been used to investigate spindle orientation, control of Cdc42 activity has been achieved using optogenetic plasma membrane translocation of the full-length protein [Strickland et al., 2012] or GEFs, for example, the catalytic DH/ PH domain of the Cdc42 GEF ITSN [Levskaya et al., 2009]; by photoswitchable steric inhibition of the active site of Cdc42 [Wu et al., 2009]; or via upstream G protein-coupled receptor activation [Bell et al., 2021].This has provided insights into how the subcellular activity of Cdc42 drives cellular and tissue morphogenetic processes, for example, front-rear polarity during cell migration [de Beco et al., 2018], and the formation of a basal actomyosin network that drives Drosophila egg chamber elongation [Popkova et al., 2020].Quantitative control of protein signalling can  RhoA (bright green) and induces contractile ring formation and cleavage furrow formation [Wagner and Glotzer, 2016].The nucleus is depicted in navy.c Manipulating Drosophila blastoderm epithelium folding.During Drosophila gastrulation, a ventral furrow forms mediated by apical constriction of the ventral blastoderm epithelial surface.(i) Recruitment of RhoGEF2 (orange) to the apical plasma membrane of dorsal epithelium activates Rho1 signalling (green) resulting in myosin-II-dependent apical constriction and ectopic dorsal tissue invagination [Izquierdo et al., 2018].(ii) Recruitment of RhoGEF2 (orange) to the basal plasma membrane of ventral epithelium activates Rho1 signalling (green) resulting in basal actomyosin upregulation and disruption of apical constriction and ventral tissue invagination [Krueger et al., 2018].
DOI: 10.1159/000528796 also be achieved optogenetically, using digital mirror devices to create gradients of light and biosensors as a quantitative readout of the level of activation.These were used to generate quantitative gradients of ITSN1-DH/PH recruitment, and therefore gradients in Cdc42 activity, which modulated migration directionality [de Beco et al., 2018].Biosensors can also be used to validate the level of optogenetic activation to endogenous levels.

Vesicular Trafficking and Establishment of Membrane Protein Domains
Vesicular trafficking delivers membrane and proteins to enable the formation, expansion, and maintenance of specialised membrane domains, which is required during cell division and AB polarity establishment.As well as trafficking newly synthesised proteins from the trans-Golgi network, existing molecules from the plasma membrane are recycled and sorted to the appropriate domain by vesicles [Jewett and Prekeris, 2018].Rab GTPases regulate vesicle budding, identity, transport, docking and fusion through the recruitment of effectors such as coat proteins, motor proteins, and tethering proteins [Stenmark, 2009;Pfeffer, 2013].Rab GTPases, like Rho family GTPases, act as molecular switches regulated by GEFs and GAPs [Stenmark, 2009].
During cell division, the regulation of endocytosis and exocytosis is important for many of the key steps of mitosis, such as spindle orientation, the ingression of the cleavage furrow, stability of the intercellular bridge and abscission [Hehnly and Doxsey, 2014;Frémont and Echard, 2018].A key step at which the mechanisms of cell division and AB polarity converge is during cytokinesis.This requires trafficking and fusion of vesicles at the midbody, and membrane fission, mediated by the endosomal sorting complex required for transport III (ESCRT-III) [Frémont and Echard, 2018].Several Rabs, such as Rab8, Rab11, Rab14, and Rab35, are implicated in vesicle targeting to the intercellular bridge to enable both abscission and delivery of apical proteins to the midbody-associated AMIS during lumen formation (Fig. 1b) [Rodriguez-Boulan and Macara, 2014;Frémont and Echard, 2018;Jewett and Prekeris, 2018].
The exact mechanisms by which apically directed proteins are trafficked and associated with the AMIS is currently an area of active research.3D epithelial cyst culture systems have revealed much about the Rab GTPases and their effectors involved in trafficking to the AMIS.For example, during epithelial lumen morphogenesis of MDCK cyst cultures, Rab11 regulates budding, microtubule-based transport and docking of apical endosomes to the apical membrane through interactions with its effector family of Rab11-interacting protein 5 (FIP5) and sorting nexin 18, kinesin-2, and cingulin [Willenborg et al., 2011;Li et al., 2014a;Mangan et al., 2016].Rab11A is also involved in a "Rab cascade"; it recruits Rabin8, a Rab GEF, to activate Rab8A, which cooperate together to deliver vesicles to the AMIS [Bryant et al., 2010].Additionally, Rab35 at the midbody-associated AMIS tethers apical vesicles through direct binding of the cytoplasmic tail of podocalyxin, a transmembrane protein and early apical marker [Klinkert et al., 2016].Recently, it was demonstrated in Caco-2 cysts that the transmembrane aminopeptidase, CD13, associates with and promotes the loading of PAR-6 into Rab11-endosomes, and acts upstream of Rab35 accumulation [Wang et al., 2021].Therefore, there are several overlapping and interrelated molecular mechanisms for apical vesicle delivery to the AMIS, but it is not fully clear how these are coordinated during celldivision-linked AB polarity establishment.
The importance of vesicular trafficking in aligning AB polarity and cell division was recently demonstrated within the zebrafish neural tube, where a lack of functional Rab11a resulted in the lack of accumulation of Crumbs protein at the forming apical midline of the neural rod.This prevented both the proper formation of apical-lateral junctions and the separation of neural epithelial cells following mitosis, therefore abolishing lumen opening [Symonds et al., 2020].The requirement for Rab11a in abscission and lumen formation was also shown via optogenetic sequestration in the zebrafish Kupffer's vesicle [Rathbun et al., 2020].Additional optogenetic approaches will enable further investigation into the hierarchy of molecular interactions driving appropriate vesicle trafficking, which is necessary for the coordination of cell division and AB polarity establishment.

Optogenetic Control of Vesicular Trafficking and Membrane Protein Localisation
The optogenetic modulation of protein trafficking and localisation of proteins at specific membrane domains has so far been achieved via protein sequestration, hijacking of cytoskeletal motor proteins or direct recruitment of full-length proteins.The rapid sequestration of, for example, Rab GTPases allows researchers to test the precise roles of different Rab proteins at different points during trafficking (for example, endocytosis, transcytosis, membrane fusion).In addition, these approaches are useful when mutants are lethal, or tissue scale phenotypes are hard to interpret.Ectopic localisation of organelles such as Rab11-specific endosomes or proteins such as the po-Cells Tissues Organs 2024;213:161-179 DOI: 10.1159/000528796 larity-associated scaffolding protein PAR-3 provides a test of their sufficiency to recruit downstream partners and to initiate processes such as AMIS formation.Together, these approaches could allow researchers to directly unpick the complex and interrelated trafficking, signalling and protein assembly pathways leading to the establishment of specialised membrane domains during both AB polarity establishment and cell mitosis.
The optogenetic protein clustering tool LARIAT (lightactivated reversible inhibition by assembled trap) utilises both the light-induced homodimerisation of cryptochrome 2 and the light-induced heterodimerisation with its partner CIB1 to rapidly and reversibly sequester CRY2linked proteins into aggregates of CIB1-linked multimeric protein oligomers [Lee et al., 2014].A modification of this approach (LARIAT of intracellular membranes, IM-LARIAT) was used in vitro to inhibit different Rab GTPases in hippocampal cells, by inducing the sequestering of CIB1-linked Rab GTPases to cytoplasmically localised CRY2 proteins, which cluster after blue-light illumination (Fig. 4a) [Nguyen et al., 2016].This revealed differential functions of Rab5 and Rab11 in mediating growth cone protrusion and longer term stability [Nguyen et al., 2016].The functionality of the IM-LARIAT approach was demonstrated in vivo in the zebrafish Kupffer's vesicle, where the requirement of Rab11 for abscission [Skop et al., 2001;Fielding et al., 2005;Wilson et al., 2005;Pohl and Jentsch, 2008] and lumenogenesis [Buckley et al., 2013;Symonds et al., 2020] was confirmed by sequestering CIB1-linked Rab11 vesicles.Acutely inhibiting Rab11 function in this way led to formation of binucleate cells and a lack of Kupffer's vesicle lumen formation (Fig. 4) [Rathbun et al., 2020].
As well as providing a method for perturbing vesicle trafficking through sequestration of Rab GTPases, LAR-IAT-based sequestration techniques can also be used to inhibit or to generate ectopic aggregates of other proteins of interest.For example, tagging GFP-nanobodies with sequestration blocks apical vesicles trafficking to the midbody and abscission in the zebrafish Kupffer's vesicle [Rathbun et al., 2020].Optogenetic components are depicted in blue with black outlines, proteins of interest in orange, the nucleus in navy, and the midbody in orange with a black outline.Arrows depict direction of trafficking.DOI: 10.1159/000528796 LARIAT components enables the clustering of any protein that is endogenously tagged with GFP [Lee et al., 2014;Qin et al., 2017;Osswald et al., 2019;Kroll et al., 2021].This may be particularly suited for proteins whose role depends on protein clustering, such as some AB polarity complexes.For example, LARIAT-based clustering techniques have been used to investigate protein interactions between AB polarity proteins in vivo, generating conflicting results.Clustering of GFP-tagged Scribble module proteins in C. elegans intestine suggested that there were no interactions between the Scribble module proteins LET-413, DLG-1, and LGL-1 (C.elegans Scribble, Discs large, and Lethal giant larvae, respectively) [Kroll et al., 2021].However, a similar study in Drosophila follicular epithelium demonstrated that Scribble interacted with Discs large via leucine rich repeats, but neither Scribble nor Discs large coclustered with lethal giant larvae [Ventura et al., 2020].There are likely context dependent differences between these two organisms and cell types, or the differences in the duration and timing of activation may have affected the ability of these proteins to cocluster.
Several studies have demonstrated that directed port of proteins and organelles can be achieved by optogenetically induced interaction with cytoskeletal motor proteins [Duan et al., 2015;Van Bergeijk et al., 2015;Harterink et al., 2016;Adrian et al., 2017;French et al., 2017].Work from the Kapitein laboratory used the TULIP system to control the direction of transport of LOVpeptagged target organelles, such as Rab11-positive recycling endosomes, peroxisomes, and mitochondria, by optogenetic heterodimerisation to ePDZ-tagged kinesin-3, dynein, or myosin Vb [Van Bergeijk et al., 2015].In primary rat hippocampal neurons, optogenetic interaction between Rab11 and dynein at the growth cone caused a decrease in dynamics, whereas an interaction between Rab11 and kinesin caused an increase in extension, by transporting recycling endosomes away from or towards the growth cone, respectively [Van Bergeijk et al., 2015].They later validated that optogenetically directed transport functions in vivo in C. elegans neurons [Harterink et al., 2016].Therefore, in cells with highly organised microtubule cytoskeletons, such as neurons, dividing cells, or epithelial cells, optogenetic control of the direction of transport of vesicles can be used to manipulate and investigate their role in cell biological processes.Optogenetically directed transport is also possible along F-actin rich structures, such as filopodia, using myosin motors as anchors [Zhang et al., 2021].The Kapitein laboratory also demonstrated that two optogenetic systems, blue-light-mediated TULIP system and red-light-mediated phytochrome system, could be co-expressed in the same cell to independently transport target organelles depending on excitation wavelength [Adrian et al., 2017].This presents an exciting possibility to independently control positioning of multiple targets to assess their hierarchy of action.
Optogenetics can also be used to directly recruit fulllength proteins to specific membrane regions.For example, red-light-mediated phytochrome optogenetic heterodimerisation was used to ectopically recruit the scaffolding protein Pard3 (zebrafish PAR-3) in vivo to subcellular regions of neuroepithelial and enveloping layer cells of zebrafish embryos [Buckley et al., 2016;Buckley, 2019].This approach was sufficient to recruit its binding partner, Pard6 (zebrafish PAR-6), and to bias Pard3 inheritance into one daughter cell of dividing neuroepithelial cells, providing an attractive potential method to investigate the role of polarity protein inheritance in cell fate decisions [Buckley et al., 2016].However, manipulation of full-length proteins is not without challenges, especially when working in vivo.For example, in vertebrate cells, where endogenous tagging is still not simple, overexpression of polarity or trafficking-related proteins to a high level has the potential to disrupt the balance of the polarity network that is being investigated.In addition, there may be competition between optogenetically tagged proteins and both untagged wildtype protein and endogenous localisation mechanisms.As discussed for Rho GTPases above, the approach of recruiting a minimal protein such as a GEF/GAP or binding domain to the region of interest allows recruitment or activation of endogenous proteins, therefore minimising these issues.While this has not been done optogenetically yet, the specific ability of different Rab GEFs to recruit particular Rab GTPases was demonstrated by mislocalising Rab GEFs to the mitochondria via a rapamycin-induced protein heterodimerisation system [Blümer et al., 2013].Modifying this to an optogenetic heterodimerisation system should be straightforward and would provide a powerful method of localising vesicle-associated Rab GTP ases to specific membrane domains.While less straightforward, it might be possible to recruit truncated versions of other polarity and mitosis-associated proteins, providing similar minimal protein recruitment approaches.

Optogenetic Control of PIPs at the Plasma Membrane
Control of PIP composition at the cell membrane can be achieved by optogenetic localisation of a minimal protein such as a binding domain to the plasma membrane, therefore recruiting endogenous PI-kinases or PI-phosphatases to phosphorylate or dephosphorylate existing PIPs.Precise modulation of PIP biochemistry in space and time could allow the investigation into the role and hierarchy of specific PIP biosynthesis and segregation during AB polarity establishment and mitosis.
Optogenetic dephosphorylation of PI(4,5)P 2 to produce PI(4)P has been achieved by recruiting the catalytic domain of the 5-phosphatase OCRL to the plasma membrane using the Cryptochrome optogenetic system (Fig. 5b) [Idevall-Hagren et al., 2012].This method has been used to explore the roles of PI(4,5)P 2 in actomyosin and polarity protein stabilisation at the plasma membrane.Using two-photon illumination to specifically deplete PI(4,5)P 2 apically in the Drosophila blastoderm epithelium resulted in inhibited actomyosin-driven apical con-Fig. 5. Optogenetic methods for control of PI(4,5)P 2 metabolism.a Optogenetic PI(4,5)P 2 phosphorylation into PI(3,4,5)P 3 using the phytochrome system employed in Toettcher et al. [2011a].The phytochrome system consists of the phytochrome B (PHYB) photoreceptor and its interaction partner, phytochrome interacting factor (PIF), which heterodimerise upon red light illumination and dissociate upon far-red light illumination.PHYB, anchored at the plasma membrane, recruits iSH fused to PIF, to localise endogenous p110, catalytic subunit of PI3K to the plasma membrane upon red light illumination.b Optogenetic PI(4,5)P 2 dephosphorylation into PI(4)P using the cryptochrome system employed in Idevall-Hagren et al. [2012] and Guglielmi et al. [2015].The cryptochrome system consists of CRY2 and its interaction partner CIBN which heterodimerise upon blue-light illumination and dissociate in the dark.CIBN, anchored at the plasma membrane, dimerises with CRY2 fused to the catalytic domain of phosphoinositide 5-phosphatase OCRL upon blue-light illumination.Optogenetic components are depicted in blue with black outlines and proteins of interest in orange.striction and ventral furrow formation [Guglielmi et al., 2015].Optogenetic depletion of PI(4,5)P 2 in the Drosophila follicular epithelium increased cortical dynamics of basolateral polarity regulator lethal giant larvae (Lgl) at the lateral cortex, suggesting PI(4,5)P 2 binds and stabilises Lgl at the lateral membrane [Ventura et al., 2020].As well as dephosphorylating PI(4,5)P 2 , OCRL can also dephosphorylate PI(3,4,5)P 3 into PI(3,4)P 2 [Zhang et al., 1995], therefore providing a potential method to further investigate the roles of PI(3,4)P 2 and PI(4,5)P 2 in mitosis and AB polarisation.
Changes in PIP composition can be detected by using biosensors based upon PI-binding domains [Wills et al., 2018].This approach was used in combination with the Phytochrome optogenetic system, in which heterodimerisation can be actively induced and reversed with different wavelengths of light, to create a feedback control system, therefore precisely specifying the level of PI3K activation in individual cells [Toettcher et al., 2011a].The fluorescence intensity of a PI(3,4,5)P 3 biosensor (Akt-PH domain) was fed back into a computational controller to modulate the relative intensity of activating/deactivating light, and therefore the level of PI3K activation via iSH2 recruitment to the cell cortex [Toettcher et al., 2011a].This allows for more stable and precise temporal activation of an optogenetic system as well as reducing variability in activation when there is heterogeneous expression of the optogenetic components.However, biosensors for PIPs must be carefully selected as they can have affinity to multiple PIPs.For example, whilst the Akt-PH domain is typically used as a PI(3,4,5)P 3 biosensor, it also has affinity for PI(3,4)P 2 [Frech et al., 1997].

Practical Considerations
Appropriate selection of optogenetic system is important in experimental design due to their different photosensitive and biochemical properties, such as wavelength sensitivity, association/dissociation rates, requirement of exogenous cofactors, and control of reversibility [Krueger et al., 2019a].Many variables, such as expression levels and protein design, can affect the efficacy and selectivity of recruitment and therefore the phenotype from activation.Method of light delivery also impacts the specificity and efficacy of optogenetic recruitment.

Light Delivery
Light can be delivered either globally or spatially restricted within the sample.Global illumination can be achieved simply using wavelength-specific light-emitting diodes, filtered lamps, or even white light sources.If optogenetic components are expressed in a specific cell compartment or tissue, then spatial specificity can be achieved with global illumination.Patterned light sources enable further spatial specificity.Bleaching or region-of-interest functions on confocal laser scanning microscopes are commonly used for this purpose, but light intensity and scanning speed can vary between different sized regionsof-interest.Using digital mirror devices for illumination patterns allows for optogenetic recruitment independently of imaging, enabling more user control over intensity and timing [Allen, 2017;Johnson et al., 2017].The point spread function of the lasers also limits 3D specificity [Krueger and De Renzis, 2022].More precise z-resolution can be achieved using two-photon illumination [Papagiakoumou, 2013].It is also important to consider optical properties of the sample.For example, thicker specimens will scatter more light than thin transparent cells.Therefore, illumination settings need to be optimised for cell/tissue type and depth.Additionally, duration of illumination can affect the phenotype of optogenetic activation depending on the protein of interest.For example, using two pulses of 20 min of illumination with a 20 min rest compared to only a 10 min rest or 40 min of continuous illumination induced greater contraction from optogenetic RhoA activation on the cell junctions of Caco-2 epithelial monolayers [Cavanaugh et al., 2020].

Photosensitivity
Photosensitive optogenetic proteins rely on wavelength-specific light absorption by chromophores.Most blue-light systems utilise flavin chromophores that are endogenously present in mammalian cells [Losi et al., 2018].However, plant phytochromes, such as Phytochrome B, require bilin chromophores which are not present in mammalian cells [Lehtinen et al., 2022].Therefore, this must be added to culture media [Levskaya et al., 2009], injected [Buckley et al., 2016], or biosynthesised via transgenesis [Uda et al., 2020].Phytochrome-based systems are excited by red wavelengths, which present higher penetrance and lower phototoxicity than blue-light systems.Furthermore, phytochrome photoactivation is actively reversible using near-infrared light, unlike blue-light systems that passively revert to their inactive state in darkness [Losi et al., 2018;Lehtinen et al., 2022].Biological read-outs must use fluorescent markers compatible with the activation wavelength.DOI: 10.1159/000528796 Optogenetic System Development Novel and optimised optogenetic systems are continually emerging (recently reviewed [Oh et al., 2021]), which can increase their compatibility for subcellular and/or in vivo experiments.For example, the optogenetic system Magnets [Kawano et al., 2015] was engineered from Neurospora crassa LOV-domain-containing photoreceptor Vivid to generate relatively small heterodimers that are both light sensitive, enabling more precise dimerisation with less background activity.Their thermodynamic stability was then optimised for permissible temperatures for mammals [Benedetti et al., 2020].In some systems, different mutations have been engineered to modulate binding affinity and the speed of dark reversion.For example, the Kuhlman laboratory introduced mutations into the LOV domain to generate an improved dynamic range of iLID and a slow reversion time variant (sLID) and SspB was mutated to create iLID/Micro with weaker binding affinity than iLID/Nano (wildtype SspB) [Guntas et al., 2015;Zimmerman et al., 2016].
Anchor design also affects recruitment and activity.Transmembrane protein anchors, such as Stargazin, that diffuse more slowly than lipid anchors, such as the CAAX motif, enable more spatial control over subcellular recruitment [Natwick and Collins, 2021].This is particularly important to achieve spatial specificity when using nonreversible optogenetic systems.The protein structure of anchors should also be considered.For example, longer anchors may not induce expected phenotypes due to increased distance of the protein of interest from the plasma membrane [Krishnamurthy et al., 2016;Krueger et al., 2018].

Protein Expression
Optogenetic recruitment is highly sensitive to relative expression levels of anchor and bait components; higher relative membrane anchor expression improves recruitment [Toettcher et al., 2011b;Krishnamurthy et al., 2016;Natwick and Collins, 2021].Binding affinity varies between optogenetic systems and therefore expression levels must be titrated to ensure strong photoactivation with low dark-state activity [Krueger and De Renzis, 2022].Changes to optogenetic protein structure may also be needed to improve expression tolerance, for example, truncation of the C-terminus of PHYB was necessary to enable robust expression in the zebrafish embryo [Buckley et al., 2016].

Side Effects
It is important to consider possible unwanted effects, such as basal activation, constitutively active or dominant negative effects.Different optogenetic systems have different levels of basal recruitment in the dark state [Hallett et al., 2016].Different strategies for photoactivation of the same signalling pathway may also vary in efficacy and phenotypic effects.For example, Rac1 signalling can be activated by photo-uncaging a constitutively active Rac1 (called photoactivatable Rac1) [Wu et al., 2009], translocation of wildtype Rac1 to the membrane [Berlew et al., 2020], or recruitment of a GEF to the membrane to activate endogenous Rac1 [Levskaya et al., 2009].As expected, activating Rac1 signalling via translocation of the wildtype Rac1 has reportedly lower basal activity compared to photoactivatable Rac1 or translocation of constitutively active Rac1 [Berlew et al., 2020].Strategies overexpressing forms of Rac1 may perturb endogenous signalling by competing with GDP dissociation inhibitors [Goedhart and van Unen, 2019].Whilst GEF-based strategies have the benefit of activating physiological levels of signalling, they still have some basal activity [Valon et al., 2017], and their phenotypic effects have been shown to vary depending on optogenetic system and which component is tagged to the anchor or bait [Hallett et al., 2016].For example, recruiting the DH/PH domain of the Rac1 GEF, T-lymphoma invasion and metastasis-inducing protein (TIAM), to the membrane using iLID/Micro produced greater protrusions than using iLID/Nano, LOV-pep+/ePDZ or CRY2/CIBN [Hallett et al., 2016].Additionally clustering of TIAM DH/PH by homo-oligomerisation of CRY2 may even be inhibitory, as CRY2-tagged TIAM DH/PH showed less activity compared to CIBNtagged TIAM DH/PH [Hallett et al., 2016].Therefore, oligomerising optogenetic systems, such as cryptochrome [Che et al., 2015], must be used cautiously to ensure that they cause the desired effect, since some proteins-of-interest may be either activated or inhibited by clustering [Bugaj et al., 2013;Taslimi et al., 2014].

Conclusion
Several mechanisms are shared and coordinated during cell mitosis to establish AB polarity, particularly during de novo AB polarity establishment within the centre of hollowing epithelial tubes and cavities (Fig. 1).These include cytoskeletal reorganisation, directed vesicular trafficking, and changes to membrane phosphoinositide composition that mediate the formation of specialised membrane domains.There is strong evidence that directed vesicular trafficking is coordinated during cell-division-linked AMIS formation.However, the mechanisms by which cytoskeletal rearrangements Cells Tissues Organs 2024;213:161-179 DOI: 10.1159/000528796 and membrane phosphoinositide composition are coordinated in this process are less clear.Key outstanding questions include: the role of RhoA and actomyosin in apical membrane establishment; the role of polarity and junctional proteins in driving centrosome location and mitotic spindle orientation in a polarising tissue; the hierarchy of action of proteins regulating vesicular trafficking; and the coordination of PI(4,5)P 2 and PI(3,4)P 2 biosynthesis during cell division and AB polarity establishment.Optogenetic techniques to precisely manipulate specific nodes within these processes in space and time present a powerful method to better understand how mitosis and AB polarity establishment are coordinated during morphogenesis.
Significant progress has been made in the evolution of optogenetic approaches, allowing highly specific subcellular and quantitative control of a wide range of molecular mechanisms.These approaches have now started to be used in vivo, allowing the precise manipulation of, for example, actomyosin contractility [Guglielmi et al., 2015;Izquierdo et al., 2018;Shellard et al., 2018;Krueger et al., 2019bKrueger et al., , 2020;;Deneke et al., 2019;Popkova et al., 2020;Bhide et al., 2021;Herrera-Perez et al., 2021;Yamamoto et al., 2021], and the mislocalisation of polarity proteins [Buckley et al., 2016;Buckley, 2019;Ventura et al., 2020;Kroll et al., 2021] at a subcellular scale within a whole organism.The further advancement of optogenetics in vivo requires careful consideration of transgenesis approaches (to ensure that the optogenetic proteins are well expressed), and optogenetic strategy (for example, full-length protein vs. minimal activation/localisation domain).Light patterning (such as via multi-photon illumination or the use of digital mirror devices) and imaging approaches must also be adapted to allow spatially specific activation/deactivation deep within a tissue.These approaches have the potential to increase our understanding of how specific molecular mechanisms, acting at the subcellular level, affect developmental biological processes such as cell mitosis and AB polarity establishment, across scales at the organ level.

Fig. 1 .
Fig. 1.Shared mechanisms of cell division and de novo AB polarity establishment.a Cytoskeletal reorganisation.During cell division, the cytoskeleton undergoes reorganisation to form the mitotic spindle (i) and actomyosin contractile ring of the cleavage furrow (ii).(i) Domains of polar Gαi/LGN/NuMA position the spindle during metaphase.(ii) Equatorial active RhoA positions the actomyosin contractile ring during anaphase.(iii) Correct orientation and localisation of the spindle and actomyosin contractile ring is important for midbody and AB polarity placement in polarised epithelial cysts.b Vesicular and protein transport.Vesicles are trafficked to the midbody during cytokinesis (direction of trafficking indicated by arrows).This is required for abscission, and delivery of apical proteins for AMIS (orange) formation in AB polarising epithelial cells.c PIP distribution.PIPs are asymmetrically distributed in dividing cells and AB polarised epithelial cysts.In dividing cells, PI(4,5)P 2 is enriched at the cleavage furrow and PI(3,4,5) P 3 found at the poles.In epithelial cysts, PI(3,4)P 2 and PI(4,5)P 2 are enriched apically, whilst PI(3,4,5)P 3 (and to a lesser extent PI(4,5)P 2 ) is found basolaterally.

Fig. 3 .
Fig.3.Optogenetic spindle mislocalisation by engineering direct interaction of NuMA with the plasma membrane (PM).a Optogenetic NuMA-PM interaction using the LOVdomain-based iLID system employed inOkumura et al. [2018].Upon blue-light illumination, a conformational change in the AsLOV2 domain reveals SsrA (blue), which binds and recruits SspB fused to NuMA (orange).Interaction of NuMA with endogenous dynein-dynactin pulls astral microtubules to move the spindle.b (i) Sustained illumination to one pole of the cell displaces the spindle towards region of recruitment (orange).(ii) Sequential illumination away from the pole causes spindle rotation.Optogenetic components are depicted in blue with black outlines, proteins of interest in orange and DNA in navy.Arrows depict direction of spindle movement.

Fig. 2 .
Fig. 2.Optogenetic RhoA activation induces non-muscle myosin-II contractility to control cell and tissue morphology.a Optogenetic RhoA activation using the LOV-domain-based TULIP system employed inWagner and Glotzer [2016].Upon blue-light illumination, the Stargazin-membrane-protein-anchored LOVpep protein-interaction domain is unmasked and binds its interaction partner PDZ.The catalytic domain of RhoA GEF LARG (orange) fused to PDZ is recruited to the membrane and catalyses the exchange of GDP for GTP, activating RhoA (bright green) and inducing downstream non-muscle myosin-II contractility.Optogenetic components are depicted in blue with black outlines.b Generating ectopic cleavage furrows.Subcellular blue-light illumination of non-adherent interphase cells recruits LARG (orange) activating

000528796 Shared Mechanisms of AB Polarity Establishment and Mitosis and Optogenetic Techniques to Investigate Them
DOI: 10.1159/