Plus Ends Of Microtubules Research Articles

Genetic evidence for a microtubule-capture mechanism during polarised growth of Aspergillus nidulans.

The cellular switch from symmetry to polarity in eukaryotes depends on the microtubule (MT) and actin cytoskeletons. In fungi such as Schizosaccharomyces pombe or Aspergillus nidulans, the MT cytoskeleton determines the sites of actin polymerization through cortical cell-end marker proteins. Here we describe A. nidulans MT guidance protein A (MigA) as the first ortholog of the karyogamy protein Kar9 from Saccharomyces cerevisiae in filamentous fungi. A. nidulans MigA interacts with the cortical ApsA protein and is involved in spindle positioning during mitosis. MigA is also associated with septal and nuclear MT organizing centers (MTOCs). Super-resolution photoactivated localization microscopy (PALM) analyses revealed that MigA is recruited to assembling and retracting MT plus ends in an EbA-dependent manner. MigA is required for MT convergence in hyphal tips and plays a role in correct localization of the cell-end markers TeaA and TeaR. In addition, MigA interacts with a class-V myosin, suggesting that an active mechanism exists to capture MTs and to pull the ends along actin filaments. Hence, the organization of MTs and actin depend on each other, and positive feedback loops ensure robust polar growth.

PAR3 and aPKC regulate Golgi organization through CLASP2 phosphorylation to generate cell polarity.

The organization of the Golgi apparatus is essential for cell polarization and its maintenance. The polarity regulator PAR complex (PAR3, PAR6, and aPKC) plays critical roles in several processes of cell polarization. However, how the PAR complex participates in regulating the organization of the Golgi remains largely unknown. Here we demonstrate the functional cross-talk of the PAR complex with CLASP2, which is a microtubule plus-end-tracking protein and is involved in organizing the Golgi ribbon. CLASP2 directly interacted with PAR3 and was phosphorylated by aPKC. In epithelial cells, knockdown of either PAR3 or aPKC induced the aberrant accumulation of CLASP2 at the trans-Golgi network (TGN) concomitantly with disruption of the Golgi ribbon organization. The expression of a CLASP2 mutant that inhibited the PAR3-CLASP2 interaction disrupted the organization of the Golgi ribbon. CLASP2 is known to localize to the TGN through its interaction with the TGN protein GCC185. This interaction was inhibited by the aPKC-mediated phosphorylation of CLASP2. Furthermore, the nonphosphorylatable mutant enhanced the colocalization of CLASP2 with GCC185, thereby perturbing the Golgi organization. On the basis of these observations, we propose that PAR3 and aPKC control the organization of the Golgi through CLASP2 phosphorylation.

Polar electrostatic forces drive poleward chromosome motions.

Recent experiments revealing nanoscale electrostatic force generation at kinetochores for chromosome motions have prompted models for interactions between positively charged molecules in kinetochores and negative charge at and near the plus ends of microtubules. A clear picture of how kinetochores and centrosomes establish and maintain a dynamic coupling to microtubules for force generation during the complex motions of mitosis remains elusive. The molecular cell biology paradigm requires that specific molecules, or molecular geometries, for polar force generation be identified. While progress has been made regarding explanations of kinetochore-based chromosome motility, molecular machinery for chromosome poleward movements at centrosomes has yet to be identified. The present work concerns polar generation of poleward force in terms of experimentally known electric charge distributions at microtubule minus ends and centrosomes interacting over nanometer distances.

Capture of microtubule plus-ends at the actin cortex promotes axophilic neuronal migration by enhancing microtubule tension in the leading process.

Microtubules are a critical part of neuronal polarity and leading process extension, thus microtubule movement plays an important role in neuronal migration. However, the dynamics of microtubules during the forward movement of the nucleus into the leading process (nucleokinesis) is unclear and may be dependent on the cell type and mode of migration used. In particular, little is known about cytoskeletal changes during axophilic migration, commonly used in anteroposterior neuronal migration. We recently showed that leading process actin flow in migrating GnRH neurons is controlled by a signaling cascade involving IP3 receptors, CaMKK, AMPK, and RhoA. In the present study, microtubule dynamics were examined in GnRH neurons. Failure of the migration of these cells leads to the neuroendocrine disorder Kallmann Syndrome. Microtubules translocated forward along the leading process shaft during migration, but reversed direction and moved toward the nucleus when migration stalled. Blocking calcium release through IP3 receptors halted migration and induced the same reversal of microtubule translocation, while blocking cortical actin flow prevented microtubules from translocating toward the distal leading process. Super-resolution imaging revealed that microtubule plus-end tips are captured at the actin cortex through calcium-dependent mechanisms. This work shows that cortical actin flow draws the microtubule network forward through calcium-dependent capture in order to promote nucleokinesis, revealing a novel mechanism engaged by migrating neurons to facilitate movement.

STIM1 phosphorylation triggered by epidermal growth factor mediates cell migration

STIM1 is a key regulator of store-operated calcium entry (SOCE), and therefore a mediator of Ca2+ entry-dependent cellular events. Phosphorylation of STIM1 at ERK1/2 target sites has been described as enhancing STIM1 activation during intracellular Ca2+ emptying triggered by the inhibition of the sarco(endo)plasmic Ca2+-ATPase with thapsigargin. However, no physiological function is known for this specific phosphorylation. The present study examined the role of STIM1 phosphorylation in cell signaling triggered by EGF. Using a human endometrial adenocarcinoma cell line (Ishikawa cells) EGF or H-Ras(G12V), an active mutant of H-Ras, was found to trigger STIM1 phosphorylation at residues Ser575, Ser608, and Ser621, and this process was sensitive to PD0325901, an inhibitor of ERK1/2. Both, ERK1/2 activation and STIM1 phosphorylation took place in the absence of extracellular Ca2+, indicating that both events are upstream steps for Ca2+ entry activation. Also, EGF triggered the dissociation of STIM1 from EB1 (a regulator of microtubule plus-ends) in a manner similar to that reported for the activation of STIM1 by thapsigargin. Migration of the Ishikawa cells was impaired when STIM1 phosphorylation was targeted by Ser-to-Ala substitution mutation of ERK1/2 target sites. This effect was also observed with the Ca2+ channel blocker SKF96365. Phosphomimetic mutation of STIM1 restored the migration to levels similar to that found for STIM1-wild type. Finally, the increased vimentin expression and relocalization of E-cadherin triggered by EGF were largely inhibited by targeting STIM1 phosphorylation, while STIM1-S575E/S608E/S621E normalized the profiles of these two EMT markers.

Super-resolution imaging reveals that loss of the C-terminus of connexin43 limits microtubule plus-end capture and NaV1.5 localization at the intercalated disc.

It is well known that connexin43 (Cx43) forms gap junctions. We recently showed that Cx43 is also part of a protein-interacting network that regulates excitability. Cardiac-specific truncation of Cx43 C-terminus (mutant 'Cx43D378stop') led to lethal arrhythmias. Cx43D378stop localized to the intercalated disc (ID); cell-cell coupling was normal, but there was significant sodium current (INa) loss. We proposed that the microtubule plus-end is at the crux of the Cx43-INa relation. Yet, specific localization of relevant molecular players was prevented due to the resolution limit of fluorescence microscopy. Here, we use nanoscale imaging to establish: (i) the morphology of clusters formed by the microtubule plus-end tracking protein 'end-binding 1' (EB1), (ii) their position, and that of sodium channel alpha-subunit NaV1.5, relative to N-cadherin-rich sites, and (iii) the role of Cx43 C-terminus on the above-mentioned parameters and on the location-specific function of INa. Super-resolution fluorescence localization microscopy in murine adult cardiomyocytes revealed EB1 and NaV1.5 as distinct clusters preferentially localized to N-cadherin-rich sites. Extent of co-localization decreased in Cx43D378stop cells. Macropatch and scanning patch clamp showed reduced INa exclusively at cell end, without changes in unitary conductance. Experiments in Cx43-modified HL1 cells confirmed the relation between Cx43, INa, and microtubules. NaV1.5 and EB1 localization at the cell end is Cx43-dependent. Cx43 is part of a molecular complex that determines capture of the microtubule plus-end at the ID, facilitating cargo delivery. These observations link excitability and electrical coupling through a common molecular mechanism.

Eribulin disrupts EB1-microtubule plus-tip complex formation

Eribulin mesylate is a synthetic analog of halichondrin B known to bind tubulin and microtubules, specifically at their protein rich plus-ends, thereby dampening microtubule (MT) dynamics, arresting cells in mitosis, and inducing apoptosis. The proteins which bind to the MT plus-end are known as microtubule plus-end tracking proteins (+TIPs) and have been shown to promote MT growth and stabilization. Eribulin's plus-end binding suggests it may compete for binding sites with known +TIP proteins such as End-binding 1 (EB1). To better understand the impact of eribulin plus-end binding in regard to the proteins which normally bind there, cells expressing GFP-EB1 were treated with various concentrations of eribulin. In a concentration dependent manner, GFP-EB1 became dissociated from the MT plus-ends following drug addition. Similar results were found with immuno-stained fixed cells. Cells treated with low concentrations of eribulin also showed decreased ability to migrate, suggesting the decrease in MT dynamics may have a downstream effect. Extended exposure of eribulin to cells leads to total depolymerization of the MT array. Taken together, these data show eribulin effectively disrupts EB1 +TIP complex formation, providing mechanistic insights into the impact of eribulin on MT dynamics.

Acetylcholine Receptor (AChR) Clustering Is Regulated Both by Glycogen Synthase Kinase 3β (GSK3β)-dependent Phosphorylation and the Level of CLIP-associated Protein 2 (CLASP2) Mediating the Capture of Microtubule Plus-ends

The postsynaptic apparatus of the neuromuscular junction (NMJ) traps and anchors acetylcholine receptors (AChRs) at high density at the synapse. We have previously shown that microtubule (MT) capture by CLASP2, a MT plus-end-tracking protein (+TIP), increases the size and receptor density of AChR clusters at the NMJ through the delivery of AChRs and that this is regulated by a pathway involving neuronal agrin and several postsynaptic kinases, including GSK3. Phosphorylation by GSK3 has been shown to cause CLASP2 dissociation from MT ends, and nine potential phosphorylation sites for GSK3 have been mapped on CLASP2. How CLASP2 phosphorylation regulates MT capture at the NMJ and how this controls the size of AChR clusters are not yet understood. To examine this, we used myotubes cultured on agrin patches that induce AChR clustering in a two-dimensional manner. We show that expression of a CLASP2 mutant, in which the nine GSK3 target serines are mutated to alanine (CLASP2-9XS/9XA) and are resistant to GSK3β-dependent phosphorylation, promotes MT capture at clusters and increases AChR cluster size, compared with myotubes that express similar levels of wild type CLASP2 or that are noninfected. Conversely, myotubes expressing a phosphomimetic form of CLASP2 (CLASP2-8XS/D) show enrichment of immobile mutant CLASP2 in clusters, but MT capture and AChR cluster size are reduced. Taken together, our data suggest that both GSK3β-dependent phosphorylation and the level of CLASP2 play a role in the maintenance of AChR cluster size through the regulated capture and release of MT plus-ends.

Using plusTipTracker software to measure microtubule dynamics in Xenopus laevis growth cones.

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics(1,2). One of the most well-known and widely-utilized +TIPs for analyzing MT dynamics is the End-Binding protein, EB1, which binds all growing MT plus-ends, and thus, is a marker for MT polymerization(1). Many studies of EB1 behavior within growth cones have used time-consuming and biased computer-assisted, hand-tracking methods to analyze individual MTs(1-3). Our approach is to quantify global parameters of MT dynamics using the software package, plusTipTracker(4), following the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones(5). This software is a MATLAB-based, open-source, user-friendly package that combines automated detection, tracking, visualization, and analysis for movies of fluorescently-labeled +TIPs. Here, we present the protocol for using plusTipTracker for the analysis of fluorescently-labeled +TIP comets in cultured Xenopus laevis growth cones. However, this software can also be used to characterize MT dynamics in various cell types(6-8).

Mutant huntingtin affects cortical progenitor cell division and development of the mouse neocortex.

A polyglutamine expansion in huntingtin (HTT) causes the specific death of adult neurons in Huntington's disease (HD). Most studies have thus focused on mutant HTT (mHTT) toxicity in adulthood, and its developmental effects have been largely overlooked. We found that mHTT caused mitotic spindle misorientation in cultured cells by altering the localization of dynein, NuMA, and the p150(Glued) subunit of dynactin to the spindle pole and cell cortex and of CLIP170 and p150(Glued) to microtubule plus-ends. mHTT also affected spindle orientation in dividing mouse cortical progenitors, altering the thickness of the developing cortex. The serine/threonine kinase Akt, which regulates HTT function, rescued the spindle misorientation caused by the mHTT, by serine 421 (S421) phosphorylation, in cultured cells and in mice. Thus, cortical development is affected in HD, and this early defect can be rescued by HTT phosphorylation at S421.

A Critical Pull to Polarize the Cell

A Critical Pull to Polarize the Cell

Microtubule minus-end binding protein CAMSAP2 controls axon specification and dendrite development.

In neurons, most microtubules are not associated with a central microtubule-organizing center (MTOC), and therefore, both the minus and plus-ends of these non-centrosomal microtubules are found throughout the cell. Microtubule plus-ends are well established as dynamic regulatory sites in numerous processes, but the role of microtubule minus-ends has remained poorly understood. Using live-cell imaging, high-resolution microscopy, and laser-based microsurgery techniques, we show that the CAMSAP/Nezha/Patronin family protein CAMSAP2 specifically localizes to non-centrosomal microtubule minus-ends and is required for proper microtubule organization in neurons. CAMSAP2 stabilizes non-centrosomal microtubules and is required for neuronal polarity, axon specification, and dendritic branch formation in vitro and in vivo. Furthermore, we found that non-centrosomal microtubules in dendrites are largely generated by γ-Tubulin-dependent nucleation. We propose a two-step model in which γ-Tubulin initiates the formation of non-centrosomal microtubules and CAMSAP2 stabilizes the free microtubule minus-ends in order to control neuronal polarity and development.

ROS-mediated EB1 phosphorylation through Akt/GSK3β pathway: implication in cancer cell response to microtubule-targeting agents.

Microtubule-targeting agents (MTAs) are largely administered in adults and children cancers. Better deciphering their mechanism of action is of prime importance to develop more convenient therapy strategies. Here, we addressed the question of how reactive oxygen species (ROS) generation by mitochondria can be necessary for MTA efficacy. We showed for the first time that EB1 associates with microtubules in a phosphorylation-dependent manner, under control of ROS. By using phospho-defective mutants, we further characterized the Serine 155 residue as critical for EB1 accumulation at microtubule plus-ends, and both cancer cell migration and proliferation. Phosphorylation of EB1 on the Threonine 166 residue triggered opposite effects, and was identified as a requisite molecular switch in MTA activities. We then showed that GSK3β activation was responsible for MTA-triggered EB1 phosphorylation, resulting from ROS-mediated inhibition of upstream Akt. We thus disclosed here a novel pathway by which generation of mitochondrial ROS modulates microtubule dynamics through phosphorylation of EB1, improving our fundamental knowledge about this oncogenic protein, and pointing out the need to re-examine the current dogma of microtubule targeting by MTAs. The present work also provides a strong mechanistic rational to the promising therapeutic strategies that currently combine MTAs with anti-Akt targeted therapies.

Centmitor-1, a novel acridinyl-acetohydrazide, possesses similar molecular interaction field and antimitotic cellular phenotype as rigosertib, on 01910.Na.

Mitosis is an attractive target for the development of new anticancer drugs. In a search for novel mitotic inhibitors, we virtually screened for low molecular weight compounds that would possess similar steric and electrostatic features, but different chemical structure than rigosertib (ON 01910.Na), a putative inhibitor of phosphoinositide 3-kinase (PI3K) and polo-like kinase 1 (Plk1) pathways. Highest scoring hit compounds were tested in cell-based assays for their ability to induce mitotic arrest. We identified a novel acridinyl-acetohydrazide, here named as Centmitor-1 (Cent-1), that possesses highly similar molecular interaction field as rigosertib. In cells, Cent-1 phenocopied the cellular effects of rigosertib and caused mitotic arrest characterized by chromosome alignment defects, multipolar spindles, centrosome fragmentation, and activated spindle assembly checkpoint. We compared the effects of Cent-1 and rigosertib on microtubules and found that both compounds modulated microtubule plus-ends and reduced microtubule dynamics. Also, mitotic spindle forces were affected by the compounds as tension across sister kinetochores was reduced in mitotic cells. Our results showed that both Cent-1 and rigosertib target processes that occur during mitosis as they had immediate antimitotic effects when added to cells during mitosis. Analysis of Plk1 activity in cells using a Förster resonance energy transfer (FRET)-based assay indicated that neither compound affected the activity of the kinase. Taken together, these findings suggest that Cent-1 and rigosertib elicit their antimitotic effects by targeting mitotic processes without impairment of Plk1 kinase activity.

CK2 activates kinesin via induction of a conformational change.

Kinesin is the canonical plus-end microtubule motor and has been the focus of intense study since its discovery in 1985. We previously demonstrated a time-dependent inactivation of kinesin in vitro that was fully reversible by the addition of purified casein kinase 2 (CK2) and showed that this inactivation/reactivation pathway was relevant in cells. Here we show that kinesin inactivation results from a conformational change that causes the neck linker to be positioned closer to the motor domain. Furthermore, we show that treatment of kinesin with CK2 prevents and reverses this repositioning. Finally, we demonstrate that CK2 treatment facilitates ADP dissociation from the motor, resulting in a nucleotide-free state that promotes microtubule binding. Thus, we propose that kinesin inactivation results from neck-linker repositioning and that CK2-mediated reactivation results from CK2's dual ability to reverse this repositioning and to promote ADP release.

Identification of Interphase Functions for the NIMA Kinase Involving Microtubules and the ESCRT Pathway

The Never in Mitosis A (NIMA) kinase (the founding member of the Nek family of kinases) has been considered a mitotic specific kinase with nuclear restricted roles in the model fungus Aspergillus nidulans. By extending to A. nidulans the results of a synthetic lethal screen performed in Saccharomyces cerevisiae using the NIMA ortholog KIN3, we identified a conserved genetic interaction between nimA and genes encoding proteins of the Endosomal Sorting Complex Required for Transport (ESCRT) pathway. Absence of ESCRT pathway functions in combination with partial NIMA function causes enhanced cell growth defects, including an inability to maintain a single polarized dominant cell tip. These genetic insights suggest NIMA potentially has interphase functions in addition to its established mitotic functions at nuclei. We therefore generated endogenously GFP-tagged NIMA (NIMA-GFP) which was fully functional to follow its interphase locations using live cell spinning disc 4D confocal microscopy. During interphase some NIMA-GFP locates to the tips of rapidly growing cells and, when expressed ectopically, also locates to the tips of cytoplasmic microtubules, suggestive of non-nuclear interphase functions. In support of this, perturbation of NIMA function either by ectopic overexpression or through partial inactivation results in marked cell tip growth defects with excess NIMA-GFP promoting multiple growing cell tips. Ectopic NIMA-GFP was found to locate to the plus ends of microtubules in an EB1 dependent manner, while impairing NIMA function altered the dynamic localization of EB1 and the cytoplasmic microtubule network. Together, our genetic and cell biological analyses reveal novel non-nuclear interphase functions for NIMA involving microtubules and the ESCRT pathway for normal polarized fungal cell tip growth. These insights extend the roles of NIMA both spatially and temporally and indicate that this conserved protein kinase could help integrate cell cycle progression with polarized cell growth.

Kinesins Have a Dual Function in Organizing Microtubules during Both Tip Growth and Cytokinesis in Physcomitrella patens

Microtubules (MTs) play a crucial role in the anisotropic deposition of cell wall material, thereby affecting the direction of growth. A wide range of tip-growing cells display highly polarized cell growth, and MTs have been implicated in regulating directionality and expansion. However, the molecular machinery underlying MT dynamics in tip-growing plant cells remains unclear. Here, we show that highly dynamic MT bundles form cyclically in the polarized expansion zone of the moss Physcomitrella patens caulonemal cells through the coalescence of growing MT plus ends. Furthermore, the plant-specific kinesins (KINID1) that are is essential for the proper MT organization at cytokinesis also regulate the turnover of the tip MT bundles as well as the directionality and rate of cell growth. The plus ends of MTs grow toward the expansion zone, and KINID1 is necessary for the stability of a single coherent focus of MTs in the center of the zone, whose formation coincides with the accumulation of KINID1. We propose that KINID-dependent MT bundling is essential for the correct directionality of growth as well as for promoting growth per se. Our findings indicate that two localized cell wall deposition processes, tip growth and cytokinesis, previously believed to be functionally and evolutionarily distinct, share common and plant-specific MT regulatory components.

Mcp1p tracks microtubule plus ends to destabilize microtubules at cell tips

Microtubule plus ends are dynamically regulated by a wide variety of proteins for performing diverse cellular functions. Here, we show that the fission yeast Schizosaccharomyces pombe uncharacterized protein mcp1p is a microtubule plus-end tracking protein which depends on the kinesin-8 klp6p for transporting along microtubules towards microtubule plus ends. In the absence of mcp1p, microtubule catastrophe and rescue frequencies decrease, leading to an increased dwell time of microtubule plus ends at cell tips. Thus, these findings suggest that mcp1p may synergize with klp6p at microtubule plus-ends to destabilize microtubules.

An EB1-Kinesin Complex Is Sufficient to Steer Microtubule Growth In Vitro

Proper microtubule polarity underlies overall neuronal polarity, but mechanisms for maintaining microtubule polarity are not well understood. Previous live imaging in Drosophila dendritic arborization neurons showed that while microtubules are uniformly plus-end out in axons, dendrites possess uniformly minus-end-out microtubules [1]. Thus, maintaining uniform microtubule polarity in dendrites requires that growing microtubule plus ends entering branch points be actively directed toward the cell body. A model was proposed in which EB1 tracks the plus ends of microtubules growing into a branch and an associated kinesin-2 motor walks along a static microtubule to steer the plus end toward the cell body. However, the fast plus-end binding dynamics of EB1 [2-5] appear to be at odds with this proposed mechanical function. To test this model invitro, we reconstituted the system by artificially dimerizing EB1 to kinesin, growing microtubules from immobilized seeds, and imaging encounters between growing microtubule plus ends and static microtubules. Consistent with invivo observations, the EB1-kinesin complex actively steered growing microtubules. Thus, EB1 kinetics and mechanics are sufficient to bend microtubules for several seconds. Other kinesins also demonstrated thisactivity, suggesting this is a general mechanism for organizing and maintaining proper microtubule polarity in cells.

Single-molecule fluorescence and in vivo optical traps: how multiple dyneins and kinesins interact.

Kinesin and dynein walking on microtubules are the two main drivers of long-distance intracellular transport in a huge variety of systems, from neurons to melanophores. These motors, however, are oppositely directed, with (most) kinesin driving cargos towards the plus-ends of microtubules, while dynein drives cargos towards the minus-ends.1 There are only two types of dynein, cytoplasmic and axonemal, with only cytoplasmic dynein being used for organelle transport.2 In this review, when we use the term dynein, we are referring to cytoplasmic dynein. Dynein is generally associated with a large multi-subunit complex, dynactin, in vivo, which appears to be necessary for many types of transport.3 Kinesins make up a large family of motors involved in organelle transport, ranging from conventional kinesin (kinesin-1), which is a typical processive, plusended directed kinesin, to NCD, a non-processive, minus-end directed kinesin.4 In addition to dynein and kinesin, there is a third motor, myosin, which walks on actin. Oftentimes, myosin is also present on the cargo and the cargo is made to switch between microtubules and actin; the latter is often for final placement of the cargo.5 In this review we will describe experimental systems at multiple levels of complexity, including: single motor type in vitro assays, multimotor in vitro assays, purified organelle in vitro assays, and finally, in vivo cellular assays (Fig. 1). This spread of experiments allows an unprecedented view of the transport complex, as kinesin and dynein can be observed with differing components of the transport complex, i.e., different levels of accessory proteins, and in different environments. By combining measurements at all these levels of complexity, the ability to parse out the function of parts of the transport complex, and reconstitute it in vitro, becomes a real possibility. Open in a separate window Figure 1 Molecular motor interactions at different levels of complexity. A. The simplest level of complexity is a single motor with a cargo or label attached, and a microtubule track, in an in vitro environment. This has been the predominant type of experiment in the study of molecular motors. It has revealed their stepping behavior, stall force, and other characteristics. However it has little to say about motor-motor interactions. B. Complexity can be increased by adding extra motors, either multiple kinesins, dyneins or kinesin-and-dynein. This is the most basic way to study motor-motor interactions, and has been used to study the cooperativity of groups motors. Knowing the absolute number of each motor can be difficult. C. Adding in accessory proteins and parts of the transport complex, like dynactin, is the next level of complexity. How accessory proteins and signaling molecules (like cAMP or a kinase) modulate kinesin-dynein interactions can be studied in this system. D. The living cell is the most complex system in which to study motor-motor interactions. Cellular gradients, accessory proteins, Microtubule Associated Proteins (MAPs), organelles, and filament meshes are just a few of the things present that could affect transport. This complexity makes it very difficult to isolate specific causes of transport behavior, but also allows the study of motor-motor interactions in their native settings.