Membrane contacts with the endoplasmic reticulum modulate plastid morphology and behaviour.

Plastid behaviour often occurs in tandem with endoplasmic reticulum (ER) dynamics. In order to understand the underlying basis for such linked behaviour we have used time-lapse imaging-based analysis of plastid movement and pleomorphy, including the extension and retraction of stromules. Stable transgenic plants that simultaneously express fluorescent fusion proteins targeted to the plastid stroma, and the ER along with BnCLIP1-eGFP, an independent plastid envelope localized membrane contact site (MCS) marker were utilized. Our experiments strongly suggest that transient MCS formed between the plastid envelope and the ER are responsible for their concomitant behaviour.

The ER Is a Common Mediator for the Behavior and Interactions of Other Organelles.

Optimal functioning of a plant cell depends upon the efficient exchange of genetic information, ions, proteins and metabolites between the different organelles. Intuitively, increased proximity between organelles would be expected to play an important role in facilitating exchanges between them. However, it remains to be seen whether under normal, relatively non-stressed conditions organelles maintain close proximity at all. Moreover, does interactivity involve direct and frequent physical contact between the different organelles? Further, many organelles transition between spherical and tubular forms or sporadically produce thin tubular extensions, but it remains unclear whether changes in organelle morphology play a role in increasing their interactivity. Here, using targeted multicolored fluorescent fusion proteins, we report observations on the spatiotemporal relationship between plastids, mitochondria, peroxisomes and the endoplasmic reticulum in living plant cells. Under normal conditions of growth, we observe that the smaller organelles do not establish direct, physical contacts with each other but, irrespective of their individual form they all maintain intimate connectivity with the ER. Proximity between organelles does increase in response to stress through concomitant alterations in ER dynamics. Significantly, even under increased proximity the ER still remains sandwiched between the different organelles. Our observations provide strong live-imaging-based evidence for the ER acting as a common mediator in interactions between other organelles.

Study of treatment outcomes of multidrug-resistant tuberculosis under programmatic conditions and factors influencing the outcomes in Hyderabad District.

BACKGROUND: Treatment outcomes for Multidrug-Resistant Tuberculosis (MDR TB) is generally poor. The study aims to know about the treatment outcomes of MDR-TB under programmatic conditions in Hyderabad District and to analyze the factors influencing the treatment outcomes. METHODS: This is a retrospective study in which 377 patients of Hyderabad district, Telangana state who were diagnosed with MDR TB and registered at Drug Resistance TB Treatment site of Government General & Chest Hospital, Hyderabad from 4th quarter 2008 to 4th quarter 2013 were included in the study. Impact of Demographic factors (age, sex; Nutritional status (BMI); Co-morbid condition (Diabetes, HIV, Hypothyroidism); Programmatic factors (time delay in the initiation of treatment); Initial Resistance pattern on the outcomes were studied and analyzed. RESULTS: The treatment outcomes of Multidrug-Resistant Tuberculosis under Programmatic Conditions were: 57% cured, 21.8% died, 19.6% defaulted, 1.1% failed and 0.5% switched to XDR. Age, Sex, BMI had a statistically significant impact on treatment outcomes. Hypothyroidism and Delay in the initiation of treatment >1 a month had an impact on the outcomes though not statistically significant. NO impact on treatment outcomes was found when Rifampicin resistance & INH sensitive patients were compared with those resistant to both INH and Rifampicin. CONCLUSION: To reduce MDR-TB transmission in the community, improvement of treatment outcomes, via ensuring adherence, paying special attention to elderly patients is required. The Programmatic Management of Drug Resistance Tuberculosis (PMDT) should seriously think of providing Nutritional support to patients with low BMI to improve outcomes. In the programmatic conditions if we could address the problems like delay in initiation of treatment and proper management of comorbidities like HIV, Diabetes, Hypothyroidism would definitely improve the treatment outcomes.

Prospective 1-year follow-up study of all cured, new sputum smear positive tuberculosis patients under the Revised National Tuberculosis Control Program in Hyderabad, Telangana State, India.

BACKGROUND: Tuberculosis (TB) is one of the leading causes of mortality in India. The Revised National Tuberculosis Control Program (RNTCP) is a robust public health system to deal with TB in India. Unless the treated patient comes back to the system with signs and symptoms of TB due to relapse or re-infection, there is no mechanism of follow-up or any method to know the relapse rate in the population. We attempted to follow the patients declared as "Cured" as per the RNTCP guidelines for 1-2 years to identify the health status of the index cases and their household contacts in posttreatment phase. MATERIALS AND METHODS: In this prospective cohort study, 187 index cases, who were declared "Cured" in six randomly selected TB units of Hyderabad district, were followed up for 1-2 years through home visits by trained staff with structured data collection forms. Data were analyzed using SPSS v20.0. RESULTS: The mean age of the index cases was 33.64 (±16.10) years, and there were 75 females and 112 males. The study sample was homogenous for gender, age, smear grade, religion, marital status, smoking status, alcohol consumption, and human immunodeficiency virus status, etc., At 1-year posttreatment follow-up of 187 index cases, 143 (76.47%) were healthy and working without any symptoms of TB. Symptoms of TB were present in 26 (13.90%) cases, and seven index cases (4.06%) were re-diagnosed with TB. The 2-year posttreatment survival was 92%. CONCLUSION: Long-term follow-up of cured, new smear-positive TB cases reinforce the effectiveness of anti-TB treatment under the RNTCP as assessed by improved health outcomes in more than two-thirds of cases and posttreatment survival of 92% of index cases. We recommend continuing such follow-up for all TB cases treated under the RNTCP for effective end-TB strategy.

Peroxisome Mitochondria Inter-relations in Plants.

A large amount of ultrastructural, biochemical and molecular analysis indicates that peroxisomes and mitochondria not only share the same subcellular space but also maintain considerable overlap in their proteins, responses and functions. Recent approaches using imaging of fluorescent proteins targeted to both organelles in living plant cells are beginning to show the dynamic nature of their interactivity. Based on the observations of living cells, mitochondria respond rapidly to stress by undergoing fission. Mitochondrial fission is suggested to release key membrane-interacting members of the FISSION1 and DYNAMIN RELATED PROTEIN3 families and appears to be followed by the formation of thin peroxisomal extensions called peroxules. In a model we present the peroxules as an intermediate state prior to the formation of tubular peroxisomes, which, in turn are acted upon by the constriction-related proteins released by mitochondria and undergo rapid constriction and fission to increase the number of peroxisomes in a cell. The fluorescent protein aided imaging of peroxisome-mitochondria interaction provides visual evidence for their cooperation in maintenance of cellular homeostasis in plants.

Plastid Envelope-Localized Proteins Exhibit a Stochastic Spatiotemporal Relationship to Stromules.

Plastids in the viridiplantae sporadically form thin tubules called stromules that increase the interactive surface between the plastid and the surrounding cytoplasm. Several recent publications that report observations of certain proteins localizing to the extensions have then used the observations to suggest stromule-specific functions. The mechanisms by which specific localizations on these transient and sporadically formed extensions might occur remain unclear. Previous studies have yet to address the spatiotemporal relationship between a particular protein localization pattern and its distribution on an extended stromules and/or the plastid body. Here, we have used discrete protein patches found in several transgenic plants as fiducial markers to investigate this relationship. While we consider the inner plastid envelope-membrane localized protein patches of the GLUCOSE 6-PHOSPHATE/PHOSPHATE TRANSLOCATOR1 and the TRIOSE-PHOSPHATE/ PHOSPHATE TRANSLOCATOR 1 as artifacts of fluorescent fusion protein over-expression, stromule formation is not compromised in the respective stable transgenic lines that maintain normal growth and development. Our analysis of chloroplasts in the transgenic lines in the Arabidopsis Columbia background, and in the arc6 mutant, under stromule-inducing conditions shows that the possibility of finding a particular protein-enriched domain on an extended stromule or on a region of the main plastid body is stochastic. Our observations provide insights on the behavior of chloroplasts, the relationship between stromules and the plastid-body and strongly challenge claims of stromule-specific functions based solely upon protein localization to plastid extensions. ONE SENTENCE SUMMARY: Observations of the spatiotemporal relationship between plastid envelope localized fluorescent protein fusions of two sugar-phosphate transporters and stromules suggest a stochastic rather than specific localization pattern that questions the idea of independent functions for stromules.

Chloroplast behaviour and interactions with other organelles in Arabidopsis thaliana pavement cells.

Chloroplasts are a characteristic feature of green plants. Mesophyll cells possess the majority of chloroplasts and it is widely believed that, with the exception of guard cells, the epidermal layer in most higher plants does not contain chloroplasts. However, recent observations on Arabidopsis thaliana have shown a population of chloroplasts in pavement cells that are smaller than mesophyll chloroplasts and have a high stroma to grana ratio. Here, using stable transgenic lines expressing fluorescent proteins targeted to the plastid stroma, plasma membrane, endoplasmic reticulum, tonoplast, nucleus, mitochondria, peroxisomes, F-actin and microtubules, we characterize the spatiotemporal relationships between the pavement cell chloroplasts (PCCs) and their subcellular environment. Observations on the PCCs suggest a source-sink relationship between the epidermal and the mesophyll layers, and experiments with the Arabidopsis mutants glabra2 (gl2) and immutans (im), which show altered epidermal plastid development, underscored their developmental plasticity. Our findings lay down the foundation for further investigations aimed at understanding the precise role and contributions of PCCs in plant interactions with the environment.

High Light Intensity Leads to Increased Peroxule-Mitochondria Interactions in Plants.

Peroxules are thin protrusions from spherical peroxisomes produced under low levels of reactive oxygen species (ROS) stress. Whereas, stress mitigation favors peroxule retraction, prolongation of the ROS stress leads to the elongation of the peroxisome into a tubular form. Subsequently, the elongated form becomes constricted through the binding of proteins such as dynamin related proteins 3A and 3B and eventually undergoes fission to increase the peroxisomal population within a cell. The events that occur in the short time window between peroxule initiation and the tubulation of the entire peroxisome have not been observed in living plant cells. Here, using fluorescent protein aided live-imaging, we show that peroxules are formed after only 4 min of high light (HL) irradiation during which there is a perceptible increase in the cytosolic levels of hydrogen peroxide. Using a stable, double transgenic line of Arabidopsis thaliana expressing a peroxisome targeted YFP and a mitochondrial targeted GFP probe, we observed sustained interactions between peroxules and small, spherical mitochondria. Further, it was observed that the frequency of HL-induced interactions between peroxules and mitochondria increased in the Arabidopsis anisotropy1 mutant that has reduced cell wall crystallinity and where we show accumulation of higher H2O2 levels than wild type plants. Our observations suggest a testable model whereby peroxules act as interaction platforms for ROS-distressed mitochondria that may release membrane proteins and fission factors. These proteins might thus become easily available to peroxisomes and facilitate their proliferation for enhancing the ROS-combating capability of a plant cell.

Mitochondrial pleomorphy in plant cells is driven by contiguous ER dynamics.

Mitochondria are pleomorphic, double membrane-bound organelles involved in cellular energetics in all eukaryotes. Mitochondria in animal and yeast cells are typically tubular-reticulate structures and several micro-meters long but in green plants they are predominantly observed as 0.2-1.5 µm punctae. While fission and fusion, through the coordinated activity of several conserved proteins, shapes mitochondria, the endoplasmic reticulum (ER) has recently been identified as an additional player in this process in yeast and mammalian cells. The mitochondria-ER relationship in plant cells remains largely uncharacterized. Here, through live-imaging of the entire range of mitochondria pleomorphy we uncover the underlying basis for the predominantly punctate mitochondrial form in plants. We demonstrate that mitochondrial morphology changes in response to light and cytosolic sugar levels in an ER mediated manner. Whereas, large ER polygons and low dynamics under dark conditions favor mitochondrial fusion and elongation, small ER polygons result in increased fission and predominantly small mitochondria. Hypoxia also reduces ER dynamics and increases mitochondrial fusion to produce giant mitochondria. By observing elongated mitochondria in normal plants and fission-impaired Arabidopsis nmt1-2 and drp3a mutants we also establish that thin extensions called matrixules and a beads-on-a-string mitochondrial phenotype are direct consequences of mitochondria-ER interactions.

Simultaneous live-imaging of peroxisomes and the ER in plant cells suggests contiguity but no luminal continuity between the two organelles.

Transmission electron micrographs of peroxisomes in diverse organisms, including plants, suggest their close association and even luminal connectivity with the endoplasmic reticulum (ER). After several decades of debate de novo peroxisome biogenesis from the ER is strongly favored in yeasts and mammals. Unfortunately many of the proteins whose transit through the ER constitutes a major evidence for peroxisome biogenesis from the ER do not exhibit a similar localization in plants. Consequently, at best the ER acts as a membrane source for peroxisome in plants. However, in addition to their de novo biogenesis from the ER an increase in peroxisome numbers also occurs through fission of existing peroxisomes. In recent years live-imaging has been used to visualize peroxisomes and the ER but the precise spatio-temporal relationship between the two organelles has not been well-explored. Here we present our assessment of the peroxisome-ER relationship through imaging of living Arabidopsis thaliana plants simultaneously expressing different color combinations of fluorescent proteins targeted to both organelles. Our observations on double transgenic wild type and a drp3a/apm1 mutant Arabidopsis plants suggest strong correlations between the dynamic behavior of peroxisomes and the neighboring ER. Although peroxisomes and ER are closely aligned there appears to be no luminal continuity between the two. Similarly, differentially colored elongated peroxisomes of a drp3a mutant expressing a photoconvertible peroxisomal matrix protein are unable to fuse and share luminal protein despite considerable intermingling. Substantiation of our observations is suggested through 3D iso-surface rendering of image stacks, which shows closed ended peroxisomes enmeshed among ER tubules possibly through membrane contact sites (MCS). Our observations support the idea that increase in peroxisome numbers in a plant cell occurs mainly through the fission of existing peroxisomes in an ER aided manner.

Differential coloring reveals that plastids do not form networks for exchanging macromolecules.

Stroma-filled tubules named stromules are sporadic extensions of plastids. Earlier, photobleaching was used to demonstrate fluorescent protein diffusion between already interconnected plastids and formed the basis for suggesting that all plastids are able to form networks for exchanging macromolecules. However, a critical appraisal of literature shows that this conjecture is not supported by unequivocal experimental evidence. Here, using photoconvertible mEosFP, we created color differences between similar organelles that enabled us to distinguish clearly between organelle fusion and nonfusion events. Individual plastids, despite conveying a strong impression of interactivity and fusion, maintained well-defined boundaries and did not exchange fluorescent proteins. Moreover, the high pleomorphy of etioplasts from dark-grown seedlings, leucoplasts from roots, and assorted plastids in the accumulation and replication of chloroplasts5 (arc5), arc6, and phosphoglucomutase1 mutants of Arabidopsis thaliana suggested that a single plastid unit might be easily mistaken for interconnected plastids. Our observations provide succinct evidence to refute the long-standing dogma of interplastid connectivity. The ability to create and maintain a large number of unique biochemical factories in the form of singular plastids might be a key feature underlying the versatility of green plants as it provides increased internal diversity for them to combat a wide range of environmental fluctuations and stresses.

Color recovery after photoconversion of H2B::mEosFP allows detection of increased nuclear DNA content in developing plant cells.

Many higher plants are polysomatic whereby different cells possess variable amounts of nuclear DNA. The conditional triggering of endocycles results in higher nuclear DNA content (C value) that in some cases has been correlated to increased cell size. While numerous multicolored fluorescent protein (FP) probes have revealed the general behavior of the nucleus and intranuclear components, direct visualization and estimation of changes in nuclear-DNA content in live cells during their development has not been possible. Recently, monomeric Eos fluorescent protein (mEosFP) has emerged as a useful photoconvertible protein whose color changes irreversibly from a green to a red fluorescent form upon exposure to violet-blue light. The stability and irreversibility of red fluorescent mEosFP suggests that detection of green color recovery would be possible as fresh mEosFP is produced after photoconversion. Thus a ratiometric evaluation of the red and green forms of mEosFP following photoconversion could be used to estimate production of a core histone such as H2B during its concomitant synthesis with DNA in the synthesis phase of the cell cycle. Here we present proof of concept observations on transgenic tobacco (Nicotiana tabacum) Bright Yellow 2 cells and Arabidopsis (Arabidopsis thaliana) plants stably expressing H2B::mEosFP. In Arabidopsis seedlings an increase in green fluorescence is observed specifically in cells known to undergo endoreduplication. The detection of changes in nuclear DNA content by correlating color recovery of H2B::mEosFP after photoconversion is a novel approach involving a single FP. The method has potential for facilitating detailed investigations on conditions that favor increased cell size and the development of polysomaty in plants.

mEosFP-based green-to-red photoconvertible subcellular probes for plants.

Photoconvertible fluorescent proteins (FPs) are recent additions to the biologists' toolbox for understanding the living cell. Like green fluorescent protein (GFP), monomeric EosFP is bright green in color but is efficiently photoconverted into a red fluorescent form using a mild violet-blue excitation. Here, we report mEosFP-based probes that localize to the cytosol, plasma membrane invaginations, endosomes, prevacuolar vesicles, vacuoles, the endoplasmic reticulum, Golgi bodies, mitochondria, peroxisomes, and the two major cytoskeletal elements, filamentous actin and cortical microtubules. The mEosFP fusion proteins are smaller than GFP/red fluorescent protein-based probes and, as demonstrated here, provide several significant advantages for imaging of living plant cells. These include an ability to differentially color label a single cell or a group of cells in a developing organ, selectively highlight a region of a cell or a subpopulation of organelles and vesicles within a cell for tracking them, and understanding spatiotemporal aspects of interactions between similar as well as different organelles. In addition, mEosFP probes introduce a milder alternative to fluorescence recovery after photobleaching, whereby instead of photobleaching, photoconversion followed by recovery of green fluorescence can be used for estimating subcellular dynamics. Most importantly, the two fluorescent forms of mEosFP furnish bright internal controls during imaging experiments and are fully compatible with cyan fluorescent protein, GFP, yellow fluorescent protein, and red fluorescent protein fluorochromes for use in simultaneous, multicolor labeling schemes. Photoconvertible mEosFP-based subcellular probes promise to usher in a much higher degree of precision to live imaging of plant cells than has been possible so far using single-colored FPs.

A comparative study to analyze the cost of curative care at primary health center in ahmedabad.

OBJECTIVES: To determine the unit cost of curative care provided at Primary Health Centers (PHCs) and to examine the variation in unit cost in different PHCs. MATERIALS AND METHODS: The present study was carried out in three PHCs of Ahmedabad district namely Sanathal, Nandej, and Uperdal, between 1 April, 2006 and 31 March, 2007. For estimating the cost of a health program, information on all the physical and human resources that were basic inputs to the PHC services were collected and grouped into two categories, non-recurrent (capital resources vehicles, buildings, etc.) and recurrent resources (salaries, drugs, vaccines, contraceptives, maintenance, etc.). To generate the required data, two types of schedules were developed, daily time schedule and PHC/SC (Subcenter) information schedule. RESULTS: The unit cost of curative care was lowest (Rs. 29.43) for the Sanathal PHC and highest (Rs. 88.26) for the Uperdal PHC, followed by the Nandej PHC with Rs. 40.88, implying severe underutilization of curative care at the Uperdal PHC. CONCLUSIONS: Location of health facilities is a problem at many places. As relocation is not possible or even feasible, strengthening of infrastructure and facilities at these centers can be taken up immediately.

Peroxule extension over ER-defined paths constitutes a rapid subcellular response to hydroxyl stress.

Plants survive against myriad environmental odds while remaining rooted to a single spot. The time scale over which plant cells can respond to environmental cues is seldom appreciated. Fluorescent protein-assisted live imaging of peroxisomes reveals that they respond within seconds of exposure to hydrogen peroxide and hydroxyl radicals by producing dynamic extensions called peroxules. Observations of the Arabidopsis flu mutant and treatments with xenobiotics eliciting singlet oxygen and superoxide reactive oxygen species suggest that the observed responses are specific for hydroxyl radicals. Prolonged exposure to hydroxyl radicals inhibits peroxule extension, and instead causes motile and spherical peroxisomes in a cell to become immotile and elongate several-fold. Expression of photo-convertible EosFP-PTS1 demonstrates that vermiform peroxisomes result from rapid stretching of individual peroxisomes, while the subsequent 'beads-on-a-string' morphology results from differential protein distribution within an elongated tubule. Over time, the beads in elongated peroxisomes also extend peroxules randomly before undergoing asynchronous, asymmetrical fission. Peroxule extension does not appear to involve cytoskeletal elements directly, but is closely aligned with and reflects the dynamics of ER tubules. Peroxisomal responses reveal a rapidly invoked subcellular machinery that is involved in recognition of hydroxyl stress thresholds, and its possible remediation locally through extension of peroxules or globally by increasing peroxisome numbers. A matrix protein retro-flow mechanism that supports peroxisome-ER connectivity in plant cells is suggested.

Actin control over microtubules suggested by DISTORTED2 encoding the Arabidopsis ARPC2 subunit homolog.

In Arabidopsis, based on the randomly misshapen phenotype of leaf epidermal trichomes, eight genes have been grouped into a 'DISTORTED' class. Three of the DIS genes, WURM, DISTORTED1 and CROOKED have been cloned recently and encode the ARP2, ARP3 and ARPC5 subunits respectively, of a conserved actin modulating ARP2/3 complex. Here we identify a fourth gene, DISTORTED2 as the Arabidopsis homolog of the ARPC2 subunit of the ARP2/3 complex. Like other mutants in the complex dis2 trichomes also display supernumerary, randomly localized cortical actin patches. In addition dis2 trichomes possess abnormally clustered endoplasmic microtubules near sites of actin aggregation. Since microtubules are strongly implicated in the establishment and maintenance of growth directionality in higher plants our observations of aberrant microtubule clustering in dis2 trichomes suggests a convincing explanation for the randomly distorted trichome phenotype in dis mutants. In addition, the close proximity of microtubule clusters to the arbitrarily dispersed cortical actin patches in the dis mutants provides fresh insights into cytoskeletal interactions leading us to suggest that in higher plants microtubule arrangements directed towards the establishment and maintenance of polar growth-directionality are guided by cortical actin behavior and organization.

A novel localization pattern for an EB1-like protein links microtubule dynamics to endomembrane organization.

A group of microtubule-associated proteins called +TIPs (plus end tracking proteins), including EB1 family proteins, label growing microtubule ends specifically in diverse organisms and are implicated in spindle dynamics, chromosome segregation, and directing microtubules toward cortical sites. Here, we report three new EB1-like proteins from Arabidopsis and provide the intracellular localization for AtEB1, which differs from all known EB1 proteins in having a very long acidic C-terminal tail. In marked contrast to other EB1 proteins, the GFP-AtEB1 fusion protein localizes not only to microtubule plus ends but also to motile, pleiomorphic tubulovesicular membrane networks that surround other organelles and frequently merge with the endoplasmic reticulum. AtEB1 behavior thus resembles that of +TIPs, such as the cytoplasmic linker protein CLIP-170, that are known to associate with and pull along membrane tubules in animal systems but for which homologs have not been identified in plants. In addition, though EB1 proteins are believed to stabilize microtubules, a different behavior is observed for AtEB1 where instead of stabilizing a microtubule it localizes to already stabilized regions on a microtubule. The dual localization pattern of AtEB1 suggests links between microtubule plus end dynamics and endomembrane organization during polarized growth of plant cells.

Mutations in actin-related proteins 2 and 3 affect cell shape development in Arabidopsis.

ACTIN-RELATED PROTEINS 2 and 3 form the major subunits of the ARP2/3 complex, which is known as an important regulator of actin organization in diverse organisms. Here, we report that two genes, WURM and DISTORTED1, which are important for cell shape control in Arabidopsis, encode the plant ARP2 and ARP3 orthologs, respectively. Mutations in these genes result in misdirected expansion of various cell types: trichome expansion is randomized, pavement cells fail to produce lobes, hypocotyl cells curl out of the normal epidermal plane, and root hairs are sinuous. At the subcellular level, cell shape changes are linked to severe filamentous actin aggregation and compromised vacuole fusion. Because all seven subunits of the ARP2/3 complex are present in plants, our data indicate that this complex may play a pivotal role during plant cell morphogenesis.

Arabidopsis CROOKED encodes for the smallest subunit of the ARP2/3 complex and controls cell shape by region specific fine F-actin formation.

The generation of a specific cell shape requires differential growth, whereby specific regions of the cell expand more relative to others. The Arabidopsis crooked mutant exhibits aberrant cell shapes that develop because of mis-directed expansion, especially during a rapid growth phase. GFP-aided visualization of the F-actin cytoskeleton and the behavior of subcellular organelles in different cell-types in crooked and wild-type Arabidopsis revealed that localized expansion is promoted in cellular regions with fine F-actin arrays but is restricted in areas that maintain dense F-actin. This suggested that a spatiotemporal distinction between fine versus dense F-actin in a growing cell could determine the final shape of the cell. CROOKED was molecularly identified as the plant homolog of ARPC5, the smallest sub-unit of the ARP2/3 complex that in other organisms is renowned for its role in creating dendritic arrays of fine F-actin. Rescue of crooked phenotype by the human ortholog provides the first molecular evidence for the presence and functional conservation of the complex in higher plants. Our cell-biological and molecular characterization of CROOKED suggests a general actin-based mechanism for regulating differential growth and generating cell shape diversity.

Simultaneous visualization of peroxisomes and cytoskeletal elements reveals actin and not microtubule-based peroxisome motility in plants.

Peroxisomes were visualized in living plant cells using a yellow fluorescent protein tagged with a peroxisomal targeting signal consisting of the SKL motif. Simultaneous visualization of peroxisomes and microfilaments/microtubules was accomplished in onion (Allium cepa) epidermal cells transiently expressing the yellow fluorescent protein-peroxi construct, a green fluorescent protein-mTalin construct that labels filamentous-actin filaments, and a green fluorescent protein-microtubule-binding domain construct that labels microtubules. The covisualization of peroxisomes and cytoskeletal elements revealed that, contrary to the reports from animal cells, peroxisomes in plants appear to associate with actin filaments and not microtubules. That peroxisome movement is actin based was shown by pharmacological studies. For this analysis we used onion epidermal cells and various cell types of Arabidopsis including trichomes, root hairs, and root cortex cells exhibiting different modes of growth. In transient onion epidermis assay and in transgenic Arabidopsis plants, an interference with the actin cytoskeleton resulted in progressive loss of saltatory movement followed by the aggregation and a complete cessation of peroxisome motility within 30 min of drug application. Microtubule depolymerization or stabilization had no effect.